immunogold label in secretory granules that display different shapes and sizes, distinguishable only by the application of the immunoelectron microscopy technique (Fig. 14). The proportions of each lactotroph subtype fluctuate in male and female rats, with these morphological variations appearing to be associated with changes in their secretory activities [Maldonado, et al.; 1994]. In another report, we demonstrated that the presence of morphological subtypes of lactotroph cells in rat pituitary cell cultures produced different secretory responses to neuropeptides, with the type I PRL cell population showing the

Fig. 14. Immunogold labeled lactotroph subtypes.  B: Atypical lactotroph characterized by the presence of spherical electron-dense granules ranging between 100-250 nm diameters atn the upper side of the picture and other typical subtypes, with bigger mature secretory granules, seen at the bottom of figure. C: Atypical lactotroph containing weakly labeled spherical granules, exhibiting an electron dense core eccentrically placed. A typical lactotroph on the right of the figure is also seen. Bar=1 μm (Reproduced from Maldonado & Aoki, 1994; with

Classical studies on the pituitary gland performed at electron microscopy level have shown that PRL and GH are synthesized by the distinct and specific cell types, lactotrophs and somatotrophs, respectively. In addition, the application of newer techniques, with a greater resolution and sensitivity has permitted the detection of a cell that produces both PRL and GH (designated as mammosomatotroph, MS), which was able to participate as an intermediate cell in prospective functional interconversions between somatotroph and lactotroph cells in the adult pituitary gland [Beresford; 1990]. In a previous work, we reported that the colocalization of PRL and GH by double immunogold labeling in the same cell was rarely observed in the pituitary of adult rats. Moreover, oestrogen treatment, which is implicated in the transdifferentation of GH cells into PRL cells, had no effects on the MS population (Fig. 15). Thus the data obtained do not support the suggested role for MS, as transitional cells in the presumptive interconversion of PRL and GH producing cells

In our laboratory, it has been demonstrated that the various endocrine cells constituting the pituitary gland do not occur at fixed proportions and undergo extensive changes depending on the physiological and experimental conditions. Each type of cell proliferates in response to sustained stimuli induced by trophic factors and hypothalamic hormones, with this situation being reversed by a degeneration of surplus cells after the interruption of a specific

highest response to angiotensin II and TRH action [De Paul, et al.; 1997].

permission from Biocell).

[Pasolli, et al.; 1994].

Fig. 15. Mammosomatotroph cell double immunogold labeled in pituitary rat. Colocalization of growth hormone (20 nm-gold particles) and prolactin (5 nm-gold particles) in a mammosomatotroph cell (arrow). A portion of a somatotroph labeled only for growth hormone (asterisk) is at the bottom of the figure. Bar=0.25 μm. (Reproduced from Pasolli et al., 1994; with permission from Histochemistry).

stimulus [Haggi, et al.; 1986; Pasolli, et al.; 1992; Torres, et al.; 1995; Bonaterra, et al.; 1998]. Even though the involuted pituitary cells can be recognized by just using conventional electron microscopy, specific immunogold labeling of their hormonal secretory content allows the cell type involved to be identified with complete confidence (Fig. 16). Also, the presence of an increased number of dead cells activated the phagocytosis of the cell remnants and debris through the stellate cells immunolabeled to S-100 protein [Orgnero, et al.; 1993].

Fig. 16. Immunoelectron microscopy of somatotroph cells in different functional states. Two immunogold labeled somatotrophs in a section from pituitary male rats. A degenerated somatotroph can be seen in the centre and an active growth hormone positive cell on the left, both containing characteristic spherical secretory granules immunolabeled with specific antiserum. On the right, an unlabeled cell serves as the negative control. Bar=1 μm (Reproduced from Torres et al., 1995; with permission from Histochem J).

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes 89

that the specific subcellular targeting of PKC and PKC is indicative of the role of these

Bearing in mind that different PKC isozymes are implicated in modulating almost all aspects of pituitary tumorigenesis, it is important to explore the participation of these kinases in cell proliferation, survival and cell death. In contrast with the role of PKC and PKCε in promoting pituitary cell growth, it has been demonstrated in GH3B6 pituitary adenoma cells that PKCδ activation plays an crucial role in programmed cell death [Leverrier, et al.; 2002]. Also, in a recent study, we identified the non-apoptotic mechanism parapoptosis as being the predominant cell death type involved in the regression of

In tumoral pituitary cells, we determined the fine localization of PKCδ by means of immuno-electron microscopy and observed immunolabeling in the cytosolic matrix, which was associated with RER and some isolated mitochondria in intact male rats. Furthermore, bromocriptine treatment was able to enhance the immunogold labeling to PKCδ in the nuclear compartment of pituitary cells at different involutive and dying stages (Fig. 17D). These results are indicative of PKCδ intracellular translocation, inferring that a dynamic

Fig. 17. Immuno-electron labeling for PKC isozymes in pituitary cells. Immunogold particles for PKC attached to the plasma membrane (A) and PKC associated with nuclear envelope (B) (arrowhead) after mitogenic stimulation in pituitary GH3B6 tumoral cells. PKC was mainly localized to the Golgi complex (GC) (arrowhead) in proliferating lactotroph cells (C).

Bc treatment enhanced PKC labeling in the nuclear compartment associated with euchromatin and nuclear envelope (arrowhead). N: nucleus; g: granules. Bar=1 μm. (Reproduced from Petiti et al., with permission from Mol Cell Endocrinol 2008 and J Mol Histol 2009, and Palmeri et al., with permission from Toxicol Appl Pharmacol 2009).

enzymes in the regulation of the activity of normal and tumoral lactotroph cells.

pituitary tumors after bromocriptine treatment.

process occurs in pituitary cell death [Palmeri, et al.; 2009].

The size of the different pituitary cell populations was assessed by morphometric analyses of the specific immunostaining of each endocrine cell type, using light microscopy immunocytochemistry by applying immunogold complexes followed by silver enhancement. A comparative analysis of the variations in each pituitary cell population from rats submitted to different experimental and physiological conditions, revealed marked fluctuations in the size of their cell population, which was closely related to the levels of secretory activity. The response of endocrine cells exposed to increased hormonal demands was expressed not only by a release of secretory granules but also by hypertrophy and hyperplasia occurring in proportion to the levels of stimulation. In contrast, cessation of the stimulus activated regressive processes that restored the various pituitary cell populations to their initial values.

Summing up, the evaluation of the pituitary cell types by immunocytochemistry correlated with their ultrastructural changes and the serum hormone levels, which permit the metabolic cell activities and the homeostatic regulation of pituitary gland to be inferred.

#### **3.2 Subcellular translocation of kinases evidenced by immunoelectron microscopy is indicative of biological cell activities**

In order to study the mechanisms involved in modulating the proliferation and cell death of normal and tumoral pituitary cells, we investigated the intracellular translocation of protein mediators which are activated in response to different stimuli. It is well known that variations in the intracellular localization of proteins are related to their different biological functions, thus revealing dynamic processes in the cells. Immunoelectron microscopy is a key technique that can be applied to identify the specific distribution of these proteins, thereby contributing to the understanding of their functions. It has been widely demonstrated that the same protein can display opposite functions, depending on its subcellular localization, with a common example of this being the family of protein kinases C (PKC) [Akita; 2002]. These are normally inactive in the cytosolic, but once activated translocate to different cellular compartments.

In our laboratory, we investigated the regulation of cell proliferation by specific PKC isozyme translocation in normal and tumoral pituitary cells. This dynamic process was studied by using an ultrastructural immunocytochemical technique with post-embedding immunogold labeling, following protocols described previously [Petiti, et al.; 2009]. Immunoelectron microscopy demonstrated that in unstimulated normal lactotroph cells, PKC and PKC were evenly distributed throughout the cytosol and nucleus, but were not associated with any specific organelles. Interestingly, after a mitogenic treatment, the PRL cell population showed an association of gold particles, thus indicating the presence of PKC at the plasma membrane, while PKC was mainly localized to the rough endoplasmic reticulum (RER) and Golgi networks [Petiti, et al.; 2008].

In tumoral pituitary GH3B6 cells, the proliferative stimulus induced the specific translocation of both PKCs to the plasma and nuclear membrane. These results indicate the existence of a close correlation between the subcellular localization of PKC isozymes with different biological functions, revealing that the localization in the plasma and nuclear membranes is associated with cell proliferation and that their immunolabeling in Golgi networks might be linked with vesicular trafficking (Fig. 17A-C) [Petiti, et al.; 2009]. Our investigation illustrates

The size of the different pituitary cell populations was assessed by morphometric analyses of the specific immunostaining of each endocrine cell type, using light microscopy immunocytochemistry by applying immunogold complexes followed by silver enhancement. A comparative analysis of the variations in each pituitary cell population from rats submitted to different experimental and physiological conditions, revealed marked fluctuations in the size of their cell population, which was closely related to the levels of secretory activity. The response of endocrine cells exposed to increased hormonal demands was expressed not only by a release of secretory granules but also by hypertrophy and hyperplasia occurring in proportion to the levels of stimulation. In contrast, cessation of the stimulus activated regressive processes that restored the various pituitary cell

Summing up, the evaluation of the pituitary cell types by immunocytochemistry correlated with their ultrastructural changes and the serum hormone levels, which permit the metabolic cell activities and the homeostatic regulation of pituitary gland to be inferred.

**3.2 Subcellular translocation of kinases evidenced by immunoelectron microscopy is**

In order to study the mechanisms involved in modulating the proliferation and cell death of normal and tumoral pituitary cells, we investigated the intracellular translocation of protein mediators which are activated in response to different stimuli. It is well known that variations in the intracellular localization of proteins are related to their different biological functions, thus revealing dynamic processes in the cells. Immunoelectron microscopy is a key technique that can be applied to identify the specific distribution of these proteins, thereby contributing to the understanding of their functions. It has been widely demonstrated that the same protein can display opposite functions, depending on its subcellular localization, with a common example of this being the family of protein kinases C (PKC) [Akita; 2002]. These are normally inactive in the cytosolic, but once activated

In our laboratory, we investigated the regulation of cell proliferation by specific PKC isozyme translocation in normal and tumoral pituitary cells. This dynamic process was studied by using an ultrastructural immunocytochemical technique with post-embedding immunogold labeling, following protocols described previously [Petiti, et al.; 2009]. Immunoelectron microscopy demonstrated that in unstimulated normal lactotroph cells, PKC and PKC were evenly distributed throughout the cytosol and nucleus, but were not associated with any specific organelles. Interestingly, after a mitogenic treatment, the PRL cell population showed an association of gold particles, thus indicating the presence of PKC at the plasma membrane, while PKC was mainly localized to the rough endoplasmic

In tumoral pituitary GH3B6 cells, the proliferative stimulus induced the specific translocation of both PKCs to the plasma and nuclear membrane. These results indicate the existence of a close correlation between the subcellular localization of PKC isozymes with different biological functions, revealing that the localization in the plasma and nuclear membranes is associated with cell proliferation and that their immunolabeling in Golgi networks might be linked with vesicular trafficking (Fig. 17A-C) [Petiti, et al.; 2009]. Our investigation illustrates

populations to their initial values.

**indicative of biological cell activities**

translocate to different cellular compartments.

reticulum (RER) and Golgi networks [Petiti, et al.; 2008].

that the specific subcellular targeting of PKC and PKC is indicative of the role of these enzymes in the regulation of the activity of normal and tumoral lactotroph cells.

Bearing in mind that different PKC isozymes are implicated in modulating almost all aspects of pituitary tumorigenesis, it is important to explore the participation of these kinases in cell proliferation, survival and cell death. In contrast with the role of PKC and PKCε in promoting pituitary cell growth, it has been demonstrated in GH3B6 pituitary adenoma cells that PKCδ activation plays an crucial role in programmed cell death [Leverrier, et al.; 2002]. Also, in a recent study, we identified the non-apoptotic mechanism parapoptosis as being the predominant cell death type involved in the regression of pituitary tumors after bromocriptine treatment.

In tumoral pituitary cells, we determined the fine localization of PKCδ by means of immuno-electron microscopy and observed immunolabeling in the cytosolic matrix, which was associated with RER and some isolated mitochondria in intact male rats. Furthermore, bromocriptine treatment was able to enhance the immunogold labeling to PKCδ in the nuclear compartment of pituitary cells at different involutive and dying stages (Fig. 17D). These results are indicative of PKCδ intracellular translocation, inferring that a dynamic process occurs in pituitary cell death [Palmeri, et al.; 2009].

Fig. 17. Immuno-electron labeling for PKC isozymes in pituitary cells. Immunogold particles for PKC attached to the plasma membrane (A) and PKC associated with nuclear envelope (B) (arrowhead) after mitogenic stimulation in pituitary GH3B6 tumoral cells. PKC was mainly localized to the Golgi complex (GC) (arrowhead) in proliferating lactotroph cells (C). Bc treatment enhanced PKC labeling in the nuclear compartment associated with euchromatin and nuclear envelope (arrowhead). N: nucleus; g: granules. Bar=1 μm. (Reproduced from Petiti et al., with permission from Mol Cell Endocrinol 2008 and J Mol Histol 2009, and Palmeri et al., with permission from Toxicol Appl Pharmacol 2009).

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes 91

cancer, it was important to verify that they were present in the stroma in the prostatitis model,

Fig. 18. Immunogold for CC16 to analyze effects of allergic inflammation on bronchiolar Clara cell secretions at ultrastructural level. A: Normal bronquiolar Clara cells with numerous mitochondria (Mi) and few small secretory granules with round profiles and moderate electron density. B: By immunogold labeling, the protein CC16 was shown to be intense at the secretory granules, while it was moderate in the cytoplasm. C: After acute OVA challenge, Clara cells exhibit numerous large secretory granules filling the whole apical cytoplasm. D: Large and electron- lucent secretory granules, appearing after acute OVA challenge, are heavily decorated for CC16 protein. Also, free cytoplasmic labeling appears around the granules. E: After chronic OVA, mucous transformed Clara cells show characteristic large fused electron-lucent granules. F: Mucous transformed Clara cells exhibit scarce CC16 label restricted to a few positive small granules under the plasma membrane with the cytoplasm portions intermingled. CC: ciliated cell; Nu: nucleus; OVA: ovalbumin. Bar=1 µm (Reproduced from Roth et al., 2007; with permission from Histochem Cell Biol).

which was carried out by applying immunogold labeling to ACTA2 (Fig. 19F).

#### **3.3 Immunoelectron microscopy emerges as an indispensable tool to understand the differentiation status of the cell**

Diverse changes in the cell microenvironment, such as those generated by inflammatory stimuli, can induce significant alterations in both the epithelial and stromal cells, thus leading to modifications in cell biology and function. However, immunogold labeling at the ultrastructural level is necessary in order to reveal and characterize these processes, by evaluating cytoskeleton components, secretory products, growth factor receptors, etc.

In lung epithelium, we have reported changes induced by allergy inflammation in bronquiolar Clara cells (which are strongly involved in several key homeostatic mechanisms). The apical cytoplasm of these polarized secretory cells is filled with polymorphic mitochondria and scarce secretory granules under the plasma membrane (Fig. 18A). Nevertheless, even under normal conditions, the immunogold technique was able to differentiate between these two organelles: mitochondria stained positive to CYP1E2 (P450 cytochrome), while the secretory granules were easily identified by their immunoreactivity for CC16, the main secretory protein of Clara cells (Fig. 18B). After applying a short allergic stimulus, these cells hypertrophied and filled up with big secretory-like granules of moderate electron density (Fig. 18C). Then, immunogold helped us to identify the mitochondria as scarce CYP1E2-positive structures intermingled among the granules and to observe that the numerous secretory granules strongly gold-labeled for CC16 (Fig. 18D). Following chronic allergic exposition, the cells continued to be hypertrophied, but their secretory granules were bigger and fused with very low electron-densities (Fig. 18E), with immunogold indicating that they were not storage sites for CC16 (Fig. 18F). In this way, immunoelectron microscopy confirmed that a short allergy stimulus induced Clara cells to respond with hypersecreting CC16 as a protective mechanism against inflammation, while a chronic inflammatory microenvironment caused their transdifferentiation to mucous secreting cells that lost their CC16 storage in secretory granules [Roth, et al.; 2007].

Stromal cells are other important targets of microenvironmental modifications. In particular, fibroblasts and smooth muscle cells have proved to be very sensitive to cytokines and growth factors, with the transforming growth factor beta being a well-known stromal remodeling agent. A clear example of this remodeling was observed in prostatic smooth muscle cells after inflammation; under normal conditions. These cells are fully differentiated to a contractile phenotype containing mainly cytoskeleton components, as can be shown by immunolabeling smooth muscle α-actin (ACTA2), and scarce synthesis organelles (Fig. 19A and B). In experimental prostatitis, we observed phenotypic changes in these cells, including an increase in the cisternae of rough endoplasmic reticulum and mitochondria and the loss of cytoskeleton fibers (Fig. 19C), with the contribution of immunogold staining in this process having been focused on confirming the muscular nature of these cells and corroborating the reduction of the ACTA2-positive cytoskeleton compartment within the cells (Fig. 19D) [Quintar, et al.; 2010].

Another important example of remodeling in response to microenvironmental changes is the transformation of fibroblasts (constant components of the stroma) into myofibroblasts by myodifferentiation, a process that could be observed by immunolabeling smooth muscle αactin at electron microscopy level. In a prostatitis model, we detected cells with morphological features of myofibroblasts, including a well-developed rough endoplasmic reticulum and signs of nuclear activation (Fig. 19E). As these cells are associated with reactive stroma in

**3.3 Immunoelectron microscopy emerges as an indispensable tool to understand the**

Diverse changes in the cell microenvironment, such as those generated by inflammatory stimuli, can induce significant alterations in both the epithelial and stromal cells, thus leading to modifications in cell biology and function. However, immunogold labeling at the ultrastructural level is necessary in order to reveal and characterize these processes, by evaluating cytoskeleton components, secretory products, growth factor receptors, etc.

In lung epithelium, we have reported changes induced by allergy inflammation in bronquiolar Clara cells (which are strongly involved in several key homeostatic mechanisms). The apical cytoplasm of these polarized secretory cells is filled with polymorphic mitochondria and scarce secretory granules under the plasma membrane (Fig. 18A). Nevertheless, even under normal conditions, the immunogold technique was able to differentiate between these two organelles: mitochondria stained positive to CYP1E2 (P450 cytochrome), while the secretory granules were easily identified by their immunoreactivity for CC16, the main secretory protein of Clara cells (Fig. 18B). After applying a short allergic stimulus, these cells hypertrophied and filled up with big secretory-like granules of moderate electron density (Fig. 18C). Then, immunogold helped us to identify the mitochondria as scarce CYP1E2-positive structures intermingled among the granules and to observe that the numerous secretory granules strongly gold-labeled for CC16 (Fig. 18D). Following chronic allergic exposition, the cells continued to be hypertrophied, but their secretory granules were bigger and fused with very low electron-densities (Fig. 18E), with immunogold indicating that they were not storage sites for CC16 (Fig. 18F). In this way, immunoelectron microscopy confirmed that a short allergy stimulus induced Clara cells to respond with hypersecreting CC16 as a protective mechanism against inflammation, while a chronic inflammatory microenvironment caused their transdifferentiation to mucous

secreting cells that lost their CC16 storage in secretory granules [Roth, et al.; 2007].

Stromal cells are other important targets of microenvironmental modifications. In particular, fibroblasts and smooth muscle cells have proved to be very sensitive to cytokines and growth factors, with the transforming growth factor beta being a well-known stromal remodeling agent. A clear example of this remodeling was observed in prostatic smooth muscle cells after inflammation; under normal conditions. These cells are fully differentiated to a contractile phenotype containing mainly cytoskeleton components, as can be shown by immunolabeling smooth muscle α-actin (ACTA2), and scarce synthesis organelles (Fig. 19A and B). In experimental prostatitis, we observed phenotypic changes in these cells, including an increase in the cisternae of rough endoplasmic reticulum and mitochondria and the loss of cytoskeleton fibers (Fig. 19C), with the contribution of immunogold staining in this process having been focused on confirming the muscular nature of these cells and corroborating the reduction of the ACTA2-positive cytoskeleton compartment within the

Another important example of remodeling in response to microenvironmental changes is the transformation of fibroblasts (constant components of the stroma) into myofibroblasts by myodifferentiation, a process that could be observed by immunolabeling smooth muscle αactin at electron microscopy level. In a prostatitis model, we detected cells with morphological features of myofibroblasts, including a well-developed rough endoplasmic reticulum and signs of nuclear activation (Fig. 19E). As these cells are associated with reactive stroma in

**differentiation status of the cell**

cells (Fig. 19D) [Quintar, et al.; 2010].

cancer, it was important to verify that they were present in the stroma in the prostatitis model, which was carried out by applying immunogold labeling to ACTA2 (Fig. 19F).

Fig. 18. Immunogold for CC16 to analyze effects of allergic inflammation on bronchiolar Clara cell secretions at ultrastructural level. A: Normal bronquiolar Clara cells with numerous mitochondria (Mi) and few small secretory granules with round profiles and moderate electron density. B: By immunogold labeling, the protein CC16 was shown to be intense at the secretory granules, while it was moderate in the cytoplasm. C: After acute OVA challenge, Clara cells exhibit numerous large secretory granules filling the whole apical cytoplasm. D: Large and electron- lucent secretory granules, appearing after acute OVA challenge, are heavily decorated for CC16 protein. Also, free cytoplasmic labeling appears around the granules. E: After chronic OVA, mucous transformed Clara cells show characteristic large fused electron-lucent granules. F: Mucous transformed Clara cells exhibit scarce CC16 label restricted to a few positive small granules under the plasma membrane with the cytoplasm portions intermingled. CC: ciliated cell; Nu: nucleus; OVA: ovalbumin. Bar=1 µm (Reproduced from Roth et al., 2007; with permission from Histochem Cell Biol).

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes 93

This review was written with the main purpose of emphasizing the great potentiality of immunolabeling as an indispensable tool to combine the ultrastructure with the specific identification of the molecules involved in biological processes, thus contributing to the understanding of the structure–function relationships. In order to encourage scientists from different fields of cell biology, the challenge of immunocytochemistry at ultrastructural level has been treated using a wide approach, thereby providing the knowledge and criteria necessary to implement various techniques and to suggest solution the problems inherent to this methodology. Studies performed by our research group have been described, with the identification of key molecules by immunoelectron microscopy permitting dynamic cell

We are especially grateful to Prof. Dr. Agustín Aoki, whose tireless dedication to the field of transmission electron microscopy has enabled us to relate, in this chapter, our experience in the immunoelectron microscopy field. Drs. Elsa Orgnero-Gaisán, Alejandro Peralta Soler, Amalia Pasolli, Ernesto Haggi, Mónica Bonaterra, Félix Roth and Claudia Palmeri are gratefully acknowledged for their generous contribution to the immunoelectron microscopy images. Also, the authors wish to thank Roald Pittau, Mrs Mercedes Guevara, Mrs Elena Pereyra and Mrs. Lucía Artino for their excellent technical assistance and native English speaker Dr. Paul Hobson for revising the English of this chapter. This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)

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**4. Conclusion**

processes in different tissues to be inferred.

**5. Acknowledgment**

**6. References**

5564

Fig. 19. Immunogold for ACTA2 to analyze the effects of prostatitis on cytoskeleton components of prostatic stromal cells at ultrastructural level. A: Periacinar smooth muscle cell (SMC) in normal prostate gland. B: ACTA2 immunogold labeling indicates a homogenously distributed actin cytoskeleton in periacinar smooth muscle cells on normal prostate gland. C: Smooth muscle cell in prostatitis. Note the large development of cytoplasmic organelles accompanied by loss of contractile filaments. D: ACTA2 immunolabeling on smooth muscle cell indicates that actin filaments are restricted to peripheral zones of these periacinar cells in prostatitis. E: In prostatitis, a periacinar stimulated fibroblast exhibits well developed rough endoplasmic reticulum; smooth muscle cells exhibit signs of dedifferentiation as described in figure C. F: ACTA2 immunolabeling exposes the myodifferentiation process occurring in stimulated periacinar fibroblast in prostatitis. Nu: nucleus; Fi: fibroblast; EC: epithelial cell. Bar=1 µm. (Reproduced from Quintar et al., 2010; with permission from Prostate).

## **4. Conclusion**

92 Applications of Immunocytochemistry

Fig. 19. Immunogold for ACTA2 to analyze the effects of prostatitis on cytoskeleton components of prostatic stromal cells at ultrastructural level. A: Periacinar smooth muscle

homogenously distributed actin cytoskeleton in periacinar smooth muscle cells on normal prostate gland. C: Smooth muscle cell in prostatitis. Note the large development of cytoplasmic organelles accompanied by loss of contractile filaments. D: ACTA2 immunolabeling on smooth muscle cell indicates that actin filaments are restricted to peripheral zones of these periacinar cells in prostatitis. E: In prostatitis, a periacinar

stimulated fibroblast exhibits well developed rough endoplasmic reticulum; smooth muscle cells exhibit signs of dedifferentiation as described in figure C. F: ACTA2 immunolabeling exposes the myodifferentiation process occurring in stimulated periacinar fibroblast in prostatitis. Nu: nucleus; Fi: fibroblast; EC: epithelial cell. Bar=1 µm. (Reproduced from

cell (SMC) in normal prostate gland. B: ACTA2 immunogold labeling indicates a

Quintar et al., 2010; with permission from Prostate).

This review was written with the main purpose of emphasizing the great potentiality of immunolabeling as an indispensable tool to combine the ultrastructure with the specific identification of the molecules involved in biological processes, thus contributing to the understanding of the structure–function relationships. In order to encourage scientists from different fields of cell biology, the challenge of immunocytochemistry at ultrastructural level has been treated using a wide approach, thereby providing the knowledge and criteria necessary to implement various techniques and to suggest solution the problems inherent to this methodology. Studies performed by our research group have been described, with the identification of key molecules by immunoelectron microscopy permitting dynamic cell processes in different tissues to be inferred.

### **5. Acknowledgment**

We are especially grateful to Prof. Dr. Agustín Aoki, whose tireless dedication to the field of transmission electron microscopy has enabled us to relate, in this chapter, our experience in the immunoelectron microscopy field. Drs. Elsa Orgnero-Gaisán, Alejandro Peralta Soler, Amalia Pasolli, Ernesto Haggi, Mónica Bonaterra, Félix Roth and Claudia Palmeri are gratefully acknowledged for their generous contribution to the immunoelectron microscopy images. Also, the authors wish to thank Roald Pittau, Mrs Mercedes Guevara, Mrs Elena Pereyra and Mrs. Lucía Artino for their excellent technical assistance and native English speaker Dr. Paul Hobson for revising the English of this chapter. This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Fondo para la Investigación Científica y Tecnológica (FONCyT-ANPCyT).

#### **6. References**

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Maldonado, C. & Aoki, A. (1986-b). Improvement of prolactin immunolabelling in osmium-

Maldonado, C.; Saggau, W. & Forssmann, W. (1986). Cardiodilatin-immunoreactivity in

Maldonado, C. & Aoki, A. (1994). Occurrence of atypical lactotrophs associated with levels of prolactin secretory activity. *Biocell*, Vol. 18, No. 3, pp. 88-95, ISSN 0327-9545 Maldonado, C.; Castagna, L.; Rabinovich, G. & Landa C*.* (1999). Immunocytochemical study

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**5**

*Turkey*

**Immunocytochemistry of Cytoskeleton Proteins**

In this chapter, we are going to introduce some of the cytoskeleton proteins and then, discuss about the technical details and specifics of immunocytochemistry of these proteins. Immunochemistry is a powerful technique to observe tissues (immunohistochemistry) in their surroundings and inner world of cells (immunocytochemistry) in order to understand

Being the most complex life units, eukaryotic cells have an elaborate infra – structure that is composed of filamentous proteins which all together form the skeleton of cells, "cytoskeleton". The cytoskeletal proteins are not only responsible for the physical structure of the cells, but also are involved in cellular functions requiring rapid reorganization of the cytoskeletal structures. Microfilaments and microtubules are more predominantly involved in dynamic events of the cells, whereas intermediate filaments are mainly important for static structure of the cells. Both microfilaments and microtubules have control on movement within the cell. As the most prominent movement, intracellular protein trafficking is almost a continuous dynamic event, yet this protein trafficking is surpassed by more global dynamic movements of cells such as cell division, differentiation and migration processes. All of these minor and major dynamic events are orchestrated by microfilament

Microfilaments (Fig.1), also known as actin filaments, are flexible tubes composed of two – stranded helical polymers of the actin protein. Each filament has a structural polarity with a plus and minus end. Actin filaments determining the shape of the cell surface are dispersed throughout the cell, and they are necessary for whole cell locomotion, for cells to engulf

Intermediate filaments are ropelike fibers with many long strands twisted together to provide great tensile strength. They form stable dimers by wrapping around each other in a coiled – coil configuration, and their main function is to provide cells mechanical strength

and resistance to shear stress when cells are stretched (Lodish & Baltimore, 1995).

large particles by phagocytosis and to divide (Lodish & Baltimore, 1995).

lives of organisms as close as to their physiological states.

and microtubule proteins by taking turns.

**2. Cytoskeletal proteins**

**2.2 Intermediate filaments**

**2.1 Microfilaments**

**1. Introduction**

Arzu Karabay, Şirin Korulu and Ayşegül Yldz Ünal

*Istanbul Technical University*

Yi, H.; Leunissen, J.; Shi, G.; Gutekunst, C. & Hersch, S. (2001). A novel procedure for preembedding double immunogold-silver labeling at the ultrastructural level. *J Histochem Cytochem,* Vol 49, No. 3 (Mar 2001), pp.279-284, ISSN 0022-1554.

## **Immunocytochemistry of Cytoskeleton Proteins**

Arzu Karabay, Şirin Korulu and Ayşegül Yldz Ünal *Istanbul Technical University Turkey*

## **1. Introduction**

96 Applications of Immunocytochemistry

Yi, H.; Leunissen, J.; Shi, G.; Gutekunst, C. & Hersch, S. (2001). A novel procedure for pre-

*Histochem Cytochem,* Vol 49, No. 3 (Mar 2001), pp.279-284, ISSN 0022-1554.

embedding double immunogold-silver labeling at the ultrastructural level. *J*

In this chapter, we are going to introduce some of the cytoskeleton proteins and then, discuss about the technical details and specifics of immunocytochemistry of these proteins.

Immunochemistry is a powerful technique to observe tissues (immunohistochemistry) in their surroundings and inner world of cells (immunocytochemistry) in order to understand lives of organisms as close as to their physiological states.

Being the most complex life units, eukaryotic cells have an elaborate infra – structure that is composed of filamentous proteins which all together form the skeleton of cells, "cytoskeleton". The cytoskeletal proteins are not only responsible for the physical structure of the cells, but also are involved in cellular functions requiring rapid reorganization of the cytoskeletal structures. Microfilaments and microtubules are more predominantly involved in dynamic events of the cells, whereas intermediate filaments are mainly important for static structure of the cells. Both microfilaments and microtubules have control on movement within the cell. As the most prominent movement, intracellular protein trafficking is almost a continuous dynamic event, yet this protein trafficking is surpassed by more global dynamic movements of cells such as cell division, differentiation and migration processes. All of these minor and major dynamic events are orchestrated by microfilament and microtubule proteins by taking turns.

## **2. Cytoskeletal proteins**

## **2.1 Microfilaments**

Microfilaments (Fig.1), also known as actin filaments, are flexible tubes composed of two – stranded helical polymers of the actin protein. Each filament has a structural polarity with a plus and minus end. Actin filaments determining the shape of the cell surface are dispersed throughout the cell, and they are necessary for whole cell locomotion, for cells to engulf large particles by phagocytosis and to divide (Lodish & Baltimore, 1995).

#### **2.2 Intermediate filaments**

Intermediate filaments are ropelike fibers with many long strands twisted together to provide great tensile strength. They form stable dimers by wrapping around each other in a coiled – coil configuration, and their main function is to provide cells mechanical strength and resistance to shear stress when cells are stretched (Lodish & Baltimore, 1995).

Immunocytochemistry of Cytoskeleton Proteins 99

Fig. 2. Immunostaining of microtubules in fibroblast cell. Microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1). Image was

In many eukaryotic cells, minus – ends of microtubules are anchored near the centrosome, whereas the plus – ends are oriented towards the cell periphery. This so called interphase state structure of the cells is changed when the cells are committed to undergo mitosis, and this process requires reorganization of microtubules. In dividing eukaryotic cells, microtubule polymers have to be organized according to the different phases of the cell cycle during mitosis and interphase. This transition from interphase to mitosis involves both orientation reorganization and length changes of microtubule polymers. The formation of specialized mitotic spindle requires severing of relatively long microtubule polymers into

In non – dividing, terminally differentiated neurons, microtubule reorganization is much more elaborated in axonal and dendritic differentiation processes (Fig.3). In neurons, microtubules are born at the centrosomes within the cell body, and this birth place is away from their specialized functional areas such as axons and dendrites. These specialized elongated processes require non – centrosomal microtubules to maintain both the structural

obtained with laser scanning confocal microscope.

shorter pieces by a mechanism called microtubule severing.

**2.4 Microtubule severing proteins**

Fig. 1. Immunostaining of actin in fibroblast cell. Filamentous actin was labeled with Alexa Fluor 647 phalloidin. Image was obtained with laser scanning confocal microscope.

#### **2.3 Microtubules**

Among the cytoskeletal proteins, microtubules (Fig.2) are thought to have the most important roles, especially in generation of cell shape and polarity, cell division, cell growth and intracellular organelle transport.

Microtubules are polymers of – and – tubulin subunits. These subunits are arranged in a cylindrical tube of 24 nm in diameter. There are both lateral and longitudinal interactions between the tubulin heterodimer subunits. These interactions maintain the tubular form of microtubules (Lodish & Baltimore, 1995; Vale & Hartman, 1999).

In addition to – and – tubulins, there is a special third type of tubulin,  *–* tubulin. It is located in the centrosomal matrix. In animal cells, centrosomes are primary sites for microtubule nucleation, and microtubules are thought to be nucleated from  *–* tubulin ring complexes ( *–* TuRCs) within the centrosome.

Fig. 1. Immunostaining of actin in fibroblast cell. Filamentous actin was labeled with Alexa

Among the cytoskeletal proteins, microtubules (Fig.2) are thought to have the most important roles, especially in generation of cell shape and polarity, cell division, cell growth

Microtubules are polymers of – and – tubulin subunits. These subunits are arranged in a cylindrical tube of 24 nm in diameter. There are both lateral and longitudinal interactions between the tubulin heterodimer subunits. These interactions maintain the tubular form of

located in the centrosomal matrix. In animal cells, centrosomes are primary sites for

 *–* tubulin. It is

 *–* tubulin ring

Fluor 647 phalloidin. Image was obtained with laser scanning confocal microscope.

microtubules (Lodish & Baltimore, 1995; Vale & Hartman, 1999).

 *–* TuRCs) within the centrosome.

In addition to – and – tubulins, there is a special third type of tubulin,

microtubule nucleation, and microtubules are thought to be nucleated from

**2.3 Microtubules**

complexes (

and intracellular organelle transport.

Fig. 2. Immunostaining of microtubules in fibroblast cell. Microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1). Image was obtained with laser scanning confocal microscope.

#### **2.4 Microtubule severing proteins**

In many eukaryotic cells, minus – ends of microtubules are anchored near the centrosome, whereas the plus – ends are oriented towards the cell periphery. This so called interphase state structure of the cells is changed when the cells are committed to undergo mitosis, and this process requires reorganization of microtubules. In dividing eukaryotic cells, microtubule polymers have to be organized according to the different phases of the cell cycle during mitosis and interphase. This transition from interphase to mitosis involves both orientation reorganization and length changes of microtubule polymers. The formation of specialized mitotic spindle requires severing of relatively long microtubule polymers into shorter pieces by a mechanism called microtubule severing.

In non – dividing, terminally differentiated neurons, microtubule reorganization is much more elaborated in axonal and dendritic differentiation processes (Fig.3). In neurons, microtubules are born at the centrosomes within the cell body, and this birth place is away from their specialized functional areas such as axons and dendrites. These specialized elongated processes require non – centrosomal microtubules to maintain both the structural

Immunocytochemistry of Cytoskeleton Proteins 101

activities including proteolysis, protein folding, membrane trafficking, cytoskeleton regulation and organelle biogenesis (McNally & Vale, 1993; Vale, 2000). Katanin and spastin are expressed in both mitotic cells and post – mitotic neurons and they function to severe microtubules into shorter pieces to make microtubule transport and reorganization easier in

Katanin is the most well characterized microtubule – severing protein. It is a microtubule stimulated ATPase, and it forms ring structures when katanin subunits bind to adjacent

It is a heterodimer protein consisting of two subunits. 60kD (p60) enzymatic subunit carries out the ATPase and severing reactions. Other subunit, 80kD (p80), localizes katanin to the centrosome and regulates microtubule – severing activity of p60 subunit (Vale & Hartman,

N – terminal domain of p60 subunit binds microtubules and C – terminal AAA domain affects the binding affinity of the adjacent microtubule – binding domain. This stabilizes p60

N – terminal of p80 subunit is composed of WD40 repeat (proline – rich) domain and a C – terminal domain is required for dimerization with catalytic p60 subunit. Studies showed that WD40 repeat domain of p80 subunit is required for spindle pole localization of katanin. WD40 domain probably binds to another spindle pole protein (McNally, K. et al., 2000). Although p60 subunit shows its ATPase and severing activity in the absence of p80 subunit,

In immunostaining, katanin is co – localized with microtubules. In dividing cells (Fig.4), katanin is localized on the mitotic spindle towards poles that are closer to centrosome regions on mitotic apparatus throughout the cell cycle phases and in neurons (Fig.5), katanin is distributed throughout the cell body and neuronal processes to severe non – centrosomal microtubules to give rise to new axonal and dendritic branches for further

Fig. 4. Immunostaining of p60 katanin in HeLa cells. HeLa cells were labeled with 1G6 mouse p60 katanin primary antibody and visualized with Alexa Fluor 594 goat anti – mouse

IgG. Images were obtained with laser scanning confocal microscope.

tubulin subunits on the microtubule wall (McNally, F. et al., 2000).

both cell types.

**2.4.1 Katanin**

elongation.

1999; Quarmby et al., 2000).

rings (Vale & Hartman, 1999).

p80 subunit cannot sever microtubules on its own.

integrity and the activity of these terminally differentiated cells. Therefore, for axonal and dendritic differentiation, microtubules need to migrate very long distances to reach their final destinations. Recent studies support the idea that microtubule severing is also an important source of non – centrosomal microtubules of neurons (Yu et al., 2008).

Fig. 3. Immunostaining of microtubules in primary hippocampal neurons. Microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1). Image was obtained with laser scanning confocal microscope.

To fulfill the needs of both mitotic and non – mitotic cells, according to the changing conditions, microtubules need not only be regulated by their intrinsic dynamics and by some structural proteins such as MAPs (Microtubule Associated Proteins), but also by molecular motor proteins such as dynein and kinesin and microtubule – severing proteins which regulate the length of the microtubule polymers.

There are mainly two severing proteins in different types of cells, katanin and spastin. Katanin and spastin are members of a large AAA (**A**TPases **A**ssociated with various cellular **A**ctivities) protein family. This family proteins play important roles in a number of cellular activities including proteolysis, protein folding, membrane trafficking, cytoskeleton regulation and organelle biogenesis (McNally & Vale, 1993; Vale, 2000). Katanin and spastin are expressed in both mitotic cells and post – mitotic neurons and they function to severe microtubules into shorter pieces to make microtubule transport and reorganization easier in both cell types.

## **2.4.1 Katanin**

100 Applications of Immunocytochemistry

integrity and the activity of these terminally differentiated cells. Therefore, for axonal and dendritic differentiation, microtubules need to migrate very long distances to reach their final destinations. Recent studies support the idea that microtubule severing is also an

important source of non – centrosomal microtubules of neurons (Yu et al., 2008).

Fig. 3. Immunostaining of microtubules in primary hippocampal neurons. Microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone

To fulfill the needs of both mitotic and non – mitotic cells, according to the changing conditions, microtubules need not only be regulated by their intrinsic dynamics and by some structural proteins such as MAPs (Microtubule Associated Proteins), but also by molecular motor proteins such as dynein and kinesin and microtubule – severing proteins

There are mainly two severing proteins in different types of cells, katanin and spastin. Katanin and spastin are members of a large AAA (**A**TPases **A**ssociated with various cellular **A**ctivities) protein family. This family proteins play important roles in a number of cellular

TUB 2.1). Image was obtained with laser scanning confocal microscope.

which regulate the length of the microtubule polymers.

Katanin is the most well characterized microtubule – severing protein. It is a microtubule stimulated ATPase, and it forms ring structures when katanin subunits bind to adjacent tubulin subunits on the microtubule wall (McNally, F. et al., 2000).

It is a heterodimer protein consisting of two subunits. 60kD (p60) enzymatic subunit carries out the ATPase and severing reactions. Other subunit, 80kD (p80), localizes katanin to the centrosome and regulates microtubule – severing activity of p60 subunit (Vale & Hartman, 1999; Quarmby et al., 2000).

N – terminal domain of p60 subunit binds microtubules and C – terminal AAA domain affects the binding affinity of the adjacent microtubule – binding domain. This stabilizes p60 rings (Vale & Hartman, 1999).

N – terminal of p80 subunit is composed of WD40 repeat (proline – rich) domain and a C – terminal domain is required for dimerization with catalytic p60 subunit. Studies showed that WD40 repeat domain of p80 subunit is required for spindle pole localization of katanin. WD40 domain probably binds to another spindle pole protein (McNally, K. et al., 2000). Although p60 subunit shows its ATPase and severing activity in the absence of p80 subunit, p80 subunit cannot sever microtubules on its own.

In immunostaining, katanin is co – localized with microtubules. In dividing cells (Fig.4), katanin is localized on the mitotic spindle towards poles that are closer to centrosome regions on mitotic apparatus throughout the cell cycle phases and in neurons (Fig.5), katanin is distributed throughout the cell body and neuronal processes to severe non – centrosomal microtubules to give rise to new axonal and dendritic branches for further elongation.

Fig. 4. Immunostaining of p60 katanin in HeLa cells. HeLa cells were labeled with 1G6 mouse p60 katanin primary antibody and visualized with Alexa Fluor 594 goat anti – mouse IgG. Images were obtained with laser scanning confocal microscope.

Immunocytochemistry of Cytoskeleton Proteins 103

In post – mitotic neurons (Fig.7), spastin is localized in discrete nuclear domains, but most interestingly detects a specific signal in the neuritis. This signal is characteristically enriched in the distal axon and in the branching regions such as growth cones. Therefore, spastin influences microtubule dynamics in growth cones; thus regulating the stability of axons and

Fig. 7. Immunostaining of spastin in primary hippocampal neurons. Hippocampal neurons were labeled with two fluorescent dyes. GFP fused spastin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of spastin and

Among these dynamic polymers of the cytoskeleton, microtubules are the main vital elements of all eukaryotic cytoskeleton in terms of determining cellular architecture and intra – cellular movements. They achieve their functions by interacting with microtubule – related proteins. In order to analyze cellular events occurring via reorganization and dynamic behaviors of microtubules and interacting proteins in accuracy, it is essential to reveal all sub – cellular structures in great detail. Detailed pinpointing of subcellular structures requires having good cytoskeleton related antibodies with high efficiency and

Although microtubules constitute a long studied area of cell biology, and tubulin antibodies are relatively well studied, there is still a high need and demand for antibodies of microtubule interacting proteins that are more recently being studied. Also, the commercially available cytoskeleton related protein antibodies are usually polyclonal antibodies; and although these antibodies are faster and easier to obtain, they could share epitopes of different proteins, leading to non – specific binding to the other cellular proteins. In addition to this, availability of a polyclonal antibody depends on the life time of the animal. In case of the need to reproduce the same antibody, using another animal with the same antigen could end up as a failed attempt as it would be produced by generation of different clones. Therefore, using these polyclonal antibodies may sometimes be misleading in understanding the cytoskeleton related events, and this gives rise to the need to have

microtubules. Image was obtained with laser scanning confocal microscope.

monoclonal antibodies with higher specificity and as an endless supply.

axonal transport (Errico et al., 2004).

specificity.

Fig. 5. Immunostaining of microtubules in primary hippocampal neurons. Hippocampal neurons were labeled with two fluorescent dyes. GFP fused p60 katanin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of p60 katanin and microtubules (yellow). Image was obtained with laser scanning confocal microscope.

#### **2.4.2 Spastin**

Spastin is a member of AAA protein family. It belongs to the meiotic subgroup which also contains proteins involved in vesicle trafficking and microtubule dynamics. Spastin shares great homology with p60 katanin within AAA domain but they do not have homology in their N terminal region.

In dividing cells (Fig.6), spastin is mainly nuclear in interphase cells. It becomes associated with centrosomes, the spindle microtubules, the midzone and finally midbody during cell division (Errico et al., 2004).

Fig. 6. Immunostaining of spastin in neuroblastoma cells. GFP fused spastin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 405 goat anti – rabbit IgG (blue) and microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of spastin and microtubules. Image was obtained with laser scanning confocal microscope.

Fig. 5. Immunostaining of microtubules in primary hippocampal neurons. Hippocampal neurons were labeled with two fluorescent dyes. GFP fused p60 katanin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of p60 katanin and microtubules (yellow). Image was obtained with laser scanning confocal

Spastin is a member of AAA protein family. It belongs to the meiotic subgroup which also contains proteins involved in vesicle trafficking and microtubule dynamics. Spastin shares great homology with p60 katanin within AAA domain but they do not have homology in

In dividing cells (Fig.6), spastin is mainly nuclear in interphase cells. It becomes associated with centrosomes, the spindle microtubules, the midzone and finally midbody during cell

Fig. 6. Immunostaining of spastin in neuroblastoma cells. GFP fused spastin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 405 goat anti – rabbit IgG (blue) and microtubules were labeled with Cy3 Conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of spastin and microtubules. Image was obtained with laser scanning confocal

microscope.

**2.4.2 Spastin**

microscope.

their N terminal region.

division (Errico et al., 2004).

In post – mitotic neurons (Fig.7), spastin is localized in discrete nuclear domains, but most interestingly detects a specific signal in the neuritis. This signal is characteristically enriched in the distal axon and in the branching regions such as growth cones. Therefore, spastin influences microtubule dynamics in growth cones; thus regulating the stability of axons and axonal transport (Errico et al., 2004).

Among these dynamic polymers of the cytoskeleton, microtubules are the main vital elements of all eukaryotic cytoskeleton in terms of determining cellular architecture and intra – cellular movements. They achieve their functions by interacting with microtubule – related proteins. In order to analyze cellular events occurring via reorganization and dynamic behaviors of microtubules and interacting proteins in accuracy, it is essential to reveal all sub – cellular structures in great detail. Detailed pinpointing of subcellular structures requires having good cytoskeleton related antibodies with high efficiency and specificity.

Although microtubules constitute a long studied area of cell biology, and tubulin antibodies are relatively well studied, there is still a high need and demand for antibodies of microtubule interacting proteins that are more recently being studied. Also, the commercially available cytoskeleton related protein antibodies are usually polyclonal antibodies; and although these antibodies are faster and easier to obtain, they could share epitopes of different proteins, leading to non – specific binding to the other cellular proteins. In addition to this, availability of a polyclonal antibody depends on the life time of the animal. In case of the need to reproduce the same antibody, using another animal with the same antigen could end up as a failed attempt as it would be produced by generation of different clones. Therefore, using these polyclonal antibodies may sometimes be misleading in understanding the cytoskeleton related events, and this gives rise to the need to have monoclonal antibodies with higher specificity and as an endless supply.

Immunocytochemistry of Cytoskeleton Proteins 105

Fig. 9. Western blot image indicating p60 katanin in brain extract. Arrowhead points p60 katanin labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with

Fig. 10. Immunohistochemistry image showing p60 katanin expression in brain (br) tissue sections of *Gallus gallus* embryo. a) Negative control, b) p60 katanin expressing region. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with anti – mouse IgG alkaline phosphatase secondary antibody. Image was obtained with

anti – mouse IgG alkaline phosphatase secondary antibody.

light microscope.

However, obtaining a monoclonal antibody specific for a particular protein is very time consuming. Thus, there is limited source of available cytoskeleton related monoclonal antibodies for a specific protein of interest. For instance, there has been no monoclonal p60 katanin antibody available until now, and polyclonal p60 katanin antibodies have been in use (McNally & Thomas, 1998; Karabay et al., 2004). Therefore, the absence of available monoclonal antibody against p60 katanin has been an obstacle in cytoskeleton research, and our lab has recently successfully produced a mouse anti – p60 katanin monoclonal antibody, 1G6, which has been characterized in detail for many immunochemical applications and it has been proven to be a very qualitative antibody in a vast array of organisms (rat, mouse, chicken and human tested) with wide range of applications (Akkor & Karabay, 2010). Successfully obtained 1G6 recognized the endogenous p60 katanin in immunocytochemistry (Fig.8), Western blot analysis (Fig.9) and immunohistochemistry analysis (Fig.10) in addition to ELISA application.

Fig. 8. Immunostaining of p60 katanin in primary hippocampal neurons. Hippocampal neurons were labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG. Image was obtained with laser scanning confocal microscope.

However, obtaining a monoclonal antibody specific for a particular protein is very time consuming. Thus, there is limited source of available cytoskeleton related monoclonal antibodies for a specific protein of interest. For instance, there has been no monoclonal p60 katanin antibody available until now, and polyclonal p60 katanin antibodies have been in use (McNally & Thomas, 1998; Karabay et al., 2004). Therefore, the absence of available monoclonal antibody against p60 katanin has been an obstacle in cytoskeleton research, and our lab has recently successfully produced a mouse anti – p60 katanin monoclonal antibody, 1G6, which has been characterized in detail for many immunochemical applications and it has been proven to be a very qualitative antibody in a vast array of organisms (rat, mouse, chicken and human tested) with wide range of applications (Akkor & Karabay, 2010). Successfully obtained 1G6 recognized the endogenous p60 katanin in immunocytochemistry (Fig.8), Western blot analysis (Fig.9) and immunohistochemistry analysis (Fig.10) in addition

Fig. 8. Immunostaining of p60 katanin in primary hippocampal neurons. Hippocampal neurons were labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG. Image was obtained with laser scanning

to ELISA application.

confocal microscope.

Fig. 9. Western blot image indicating p60 katanin in brain extract. Arrowhead points p60 katanin labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with anti – mouse IgG alkaline phosphatase secondary antibody.

Fig. 10. Immunohistochemistry image showing p60 katanin expression in brain (br) tissue sections of *Gallus gallus* embryo. a) Negative control, b) p60 katanin expressing region. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with anti – mouse IgG alkaline phosphatase secondary antibody. Image was obtained with light microscope.

Immunocytochemistry of Cytoskeleton Proteins 107

(Fig.11, Fig.12), or to highlight the location of the specific protein (Fig.13). Furthermore, this technique even allows discriminating different types of cells in the same culture dish

If the protein of interest were to be over – expressed in cells by transfecting with the expression vector containing the sequence for the protein of interest, some cells would take up the plasmid whereas others would not. In Fig.11, 3T3 fibroblast cells that were transfected electrophoretically with Kif15 kinesin motor containing expression vector and immunostained for actin and Kif15 could be seen. This double staining would allow

Fig. 11. Immunostaining of Kif15 expressing and non – expressing fibroblast cells. GFP fused Kif15 was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and filamentous actin was labeled with Alexa Fluor 647 phalloidin (red). Image on the right panel represents merge of Kif15 and actin filaments

In Fig.12, HeLa cells that were electrophoretically transfected with GFP fused p60 katanin expression vector for over – expression of p60 katanin could be seen. The cells were immunostained with rabbit anti – GFP antibody and mouse anti – tubulin antibody. This double staining allowed discriminating the cells that were over – expressing GFP fused p60

In Fig.13, primary hippocampal neurons immunostained for cyclin A and neuron specific beta – III tubulin could be seen. The neurons were also stained with DAPI for nuclei. This triple staining would allow observing not only specifically the neurons in the culture, but

(yellow). Image was obtained with laser scanning confocal microscope.

also the exact location of cyclin A within in the neurons.

discriminating the cells that have taken up the plasmid or not.

(Fig.14).

katanin.

## **3. Production of monoclonal antibody against p60 katanin**

In our laboratory, we produced the first monoclonal antibody against p60 katanin which is named with its clone name as 1G6. Recombinant p60 katanin protein, which was produced based on a specific region of rat p60 katanin, was expressed in *Escherichia coli* and used as antigen.

p60 katanin monoclonal antibody has been produced by hybridoma technology. In hybridoma technology, hybrid cells are produced from the fusion of B cells and the myeloma cell line (hybridomas). In this technique, the idea is combining these two cells together. In case of p60 katanin hybridomas, B cells have been derived from the lymphatic tissues of recombinant p60 katanin antigen immunized animals and the myeloma cell line have brought the immortality to these B cells when they have been fused together.

The steps of hybridoma technology for 1G6 p60 katanin monoclonal antibody could be summarized briefly as followed: (Akkor & Karabay, 2010).

## **4. Immunocytochemistry of cytoskeleton and cytoskeleton related proteins**

Immunocytochemistry is a biochemical technique that applies an antibody to a specific cell protein in the cell. It can be used either to detect whether the protein exists in the sample

In our laboratory, we produced the first monoclonal antibody against p60 katanin which is named with its clone name as 1G6. Recombinant p60 katanin protein, which was produced based on a specific region of rat p60 katanin, was expressed in *Escherichia coli* and used as

p60 katanin monoclonal antibody has been produced by hybridoma technology. In hybridoma technology, hybrid cells are produced from the fusion of B cells and the myeloma cell line (hybridomas). In this technique, the idea is combining these two cells together. In case of p60 katanin hybridomas, B cells have been derived from the lymphatic tissues of recombinant p60 katanin antigen immunized animals and the myeloma cell line

The steps of hybridoma technology for 1G6 p60 katanin monoclonal antibody could be

BALB/c mice were immunized intra – peritoneally two times with 50 mg of

 After immunization, anti – recombinant p60 katanin antibody response was assayed by indirect ELISA. ELISA plates coated with recombinant p60 katanin antigenic protein were incubated with the mice sera. Anti – recombinant p60 katanin antibody binding reaction was detected by using an alkaline phosphatase conjugated anti – mouse IgG or anti – mouse polyvalent (IgA, IgM, IgG) immunoglobulins as secondary antibody. Although the monoclonal antibodies are more specific compared to polyclonal antibodies, the clones may not always be IgG type, but also IgM or IgE. In our study, we have obtained another anti – p60 katanin antibody, which is an IgM type, but that

 Mice that developed the IgG response against recombinant p60 katanin were selected for fusion studies. Polyethylenglycol (PEG) was used to fuse the two types of cells. Fused cells were cultured in Hypoxanthine Aminopterin Thymidine (HAT) selective medium for selection of hybridomas as the two types of unfused cells would die and

Hybridoma colonies that produced antibodies against desired recombinant p60 katanin

 In selected wells which had antibody response, there were more than one hybridoma colonies which would result in polyclonal response. By "limited dilution" method, the cells in antibody responding wells were dispersed to new culture plates in order to get

Hybridoma colonies that synthesize specific antibody for recombinant p60 katanin

Selected hybridomas were then produced in large quantities and antibodies were

**4. Immunocytochemistry of cytoskeleton and cytoskeleton related proteins**  Immunocytochemistry is a biochemical technique that applies an antibody to a specific cell protein in the cell. It can be used either to detect whether the protein exists in the sample

have brought the immortality to these B cells when they have been fused together.

antibody was not as specific as IgG type 1G6 anti – p60 katanin antibody.

summarized briefly as followed: (Akkor & Karabay, 2010).

recombinant p60 katanin/mouse at 2 – week intervals.

remained hybridomas continue to divide in culture plates.

antigen was selected by cross – reactivity ELISA method.

were detected by ELISA.

purified from their media.

hybrid colony produced from single cell.

**3. Production of monoclonal antibody against p60 katanin**

antigen.

(Fig.11, Fig.12), or to highlight the location of the specific protein (Fig.13). Furthermore, this technique even allows discriminating different types of cells in the same culture dish (Fig.14).

If the protein of interest were to be over – expressed in cells by transfecting with the expression vector containing the sequence for the protein of interest, some cells would take up the plasmid whereas others would not. In Fig.11, 3T3 fibroblast cells that were transfected electrophoretically with Kif15 kinesin motor containing expression vector and immunostained for actin and Kif15 could be seen. This double staining would allow discriminating the cells that have taken up the plasmid or not.

Fig. 11. Immunostaining of Kif15 expressing and non – expressing fibroblast cells. GFP fused Kif15 was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and filamentous actin was labeled with Alexa Fluor 647 phalloidin (red). Image on the right panel represents merge of Kif15 and actin filaments (yellow). Image was obtained with laser scanning confocal microscope.

In Fig.12, HeLa cells that were electrophoretically transfected with GFP fused p60 katanin expression vector for over – expression of p60 katanin could be seen. The cells were immunostained with rabbit anti – GFP antibody and mouse anti – tubulin antibody. This double staining allowed discriminating the cells that were over – expressing GFP fused p60 katanin.

In Fig.13, primary hippocampal neurons immunostained for cyclin A and neuron specific beta – III tubulin could be seen. The neurons were also stained with DAPI for nuclei. This triple staining would allow observing not only specifically the neurons in the culture, but also the exact location of cyclin A within in the neurons.

Immunocytochemistry of Cytoskeleton Proteins 109

In Fig.14, primary hippocampal neurons immunostained for Protein Kinase C (PKC) and neuron specific beta III tubulin could be seen. The neurons were also stained with DAPI for nuclei. This staining would allow discrimination of non – neuronal cells from neurons due to common anti – PKC antibody and differentiating neuron specific anti – beta III tubulin antibody. In other ways of looking, this type of immunostaining would also allow to identify the purity of primary neuron culture, in other words, contamination of neurons

Fig. 14. Immunostaining of PKC in primary hippocampal neurons. Hippocampal neurons were labeled with three fluorescent dyes. PKC was labeled with rabbit anti – PKC primary antibody and visualized with Alexa Fluor 647 goat anti – rabbit IgG (red); microtubules were labeled with mouse anti – beta III tubulin monoclonal antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG (green) and nucleus was visualized with DAPI (blue).

If single cell suspension is going to be obtained from a tissue, firstly the tissue is dissected and dissociated in Hepes – buffered, calcium – and magnesium – free Hank's balanced salt solution (HBSS), "Dissection Medium" (Table 1). Tissues and cells have to be maintained in an osmotically balanced solution at physiological pH during all stages. Following obtaining single cell suspension, cells can be plated into serum containing medium, "Plating Medium" of which the content can differ depending on the cell type (Table 2). The plating medium contains serum that provides the trace nutrients and growth factors needed for long – term growth of the cells, also amino acids and some other ingredients. Fetal Bovine Serum (FBS)

Image was obtained with laser scanning confocal microscope.

**4.1 Sample preparation and media formulations**

with non – neuronal cells.

Fig. 12. Immunostaining of p60 katanin expressing and non – expressing HeLa cells. GFP fused p60 katanin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and microtubules were labeled with Cy3 conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of p60 katanin and microtubules. Image was obtained with laser scanning confocal microscope.

Fig. 13. Immunostaining of cyclin A in primary hippocampal neurons. Hippocampal neurons were labeled with three fluorescent dyes. Cyclin A was labeled with rabbit anti – cyclin A primary antibody and visualized with Alexa Fluor 647 goat anti – rabbit IgG (red); microtubules were labeled with mouse anti – beta III tubulin monoclonal antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG (green) and nucleus was visualized with DAPI (blue). Image was obtained with laser scanning confocal microscope.

Fig. 12. Immunostaining of p60 katanin expressing and non – expressing HeLa cells. GFP fused p60 katanin was labeled with rabbit anti – GFP primary antibody and visualized with Alexa Fluor 488 goat anti – rabbit IgG (green) and microtubules were labeled with Cy3 conjugated mouse anti – beta tubulin monoclonal antibody (Clone TUB 2.1) (red). Image on the right panel represents merge of p60 katanin and microtubules. Image was obtained with

Fig. 13. Immunostaining of cyclin A in primary hippocampal neurons. Hippocampal neurons were labeled with three fluorescent dyes. Cyclin A was labeled with rabbit anti – cyclin A primary antibody and visualized with Alexa Fluor 647 goat anti – rabbit IgG (red); microtubules were labeled with mouse anti – beta III tubulin monoclonal antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG (green) and nucleus was visualized

with DAPI (blue). Image was obtained with laser scanning confocal microscope.

laser scanning confocal microscope.

In Fig.14, primary hippocampal neurons immunostained for Protein Kinase C (PKC) and neuron specific beta III tubulin could be seen. The neurons were also stained with DAPI for nuclei. This staining would allow discrimination of non – neuronal cells from neurons due to common anti – PKC antibody and differentiating neuron specific anti – beta III tubulin antibody. In other ways of looking, this type of immunostaining would also allow to identify the purity of primary neuron culture, in other words, contamination of neurons with non – neuronal cells.

Fig. 14. Immunostaining of PKC in primary hippocampal neurons. Hippocampal neurons were labeled with three fluorescent dyes. PKC was labeled with rabbit anti – PKC primary antibody and visualized with Alexa Fluor 647 goat anti – rabbit IgG (red); microtubules were labeled with mouse anti – beta III tubulin monoclonal antibody and visualized with Alexa Fluor 488 goat anti – mouse IgG (green) and nucleus was visualized with DAPI (blue). Image was obtained with laser scanning confocal microscope.

#### **4.1 Sample preparation and media formulations**

If single cell suspension is going to be obtained from a tissue, firstly the tissue is dissected and dissociated in Hepes – buffered, calcium – and magnesium – free Hank's balanced salt solution (HBSS), "Dissection Medium" (Table 1). Tissues and cells have to be maintained in an osmotically balanced solution at physiological pH during all stages. Following obtaining single cell suspension, cells can be plated into serum containing medium, "Plating Medium" of which the content can differ depending on the cell type (Table 2). The plating medium contains serum that provides the trace nutrients and growth factors needed for long – term growth of the cells, also amino acids and some other ingredients. Fetal Bovine Serum (FBS)

Immunocytochemistry of Cytoskeleton Proteins 111

Organic solvents such as methanol, ethanol, and acetone denature proteins without any covalent modifications. Their working principle is by removing bound H2O molecules of the cells. These solvents also remove membrane and some structural lipids, and cells become

For immunostaining of cytoskeletal proteins, the most sensitive and effective fixation method is with aldehydes; glutaraldehyde and paraformaldehyde. Since aldehydes fixation method requires additional permeabilization step, a detergent, Triton – X100 can be used for cell extraction that leads cell permeabilization. Triton X100 is a detergent that is mostly used to extract the membrane from cells so that the cytoskeleton can be accessed. It is usually prepared as 10% solution in ddH2O and is rotated overnight in the cold room to fully

Another buffer called "PHEM (**P**ipes – **H**epes – **E**GTA – **M**gCl2) is also necessary for microtubule stabilization. Stock solution (2X PHEM) can be prepared as indicated in the

Cell permeabilization and fixation can either be performed separately or together in one step as termed fixation/co – extraction depending on the design of the study. For instance, in our p60 katanin – microtubule co – localization studies we preferred simultaneous fixation/co – extraction method. Since p60 katanin severs microtubules into short pieces, fixing the cells first would clutter inner side of the cells with short pieces/subunits of tubulin and therefore would cause to have higher background with the antibody staining. Whereas, if the fixation/co – extraction steps were performed simultaneously, it would allow some of the tubulin subunits to be extracted from the cell and therefore would give a cleaner staining while keeping some of the short microtubule pieces inside the cells. On the other hand, if the extraction step were to be done at first, as it would cause most of the short pieces to leave the cell, it may not be possible to see short microtubule pieces that are created by katanin. Therefore, as in the case of the examples, one should decide about the methodology of the

cell permeabilization and fixation depending on the need of the question asked.

fixation of microtubules and microtubule related proteins in dividing cells.

Below you can see some examples for different cell fixation protocols commonly used in the

 To prepare paraformaldehyde fixation solution, ingredients are mixed together and pH is adjusted to 7,3 by NaOH. Then, the fixation solution needs to be heated to 60°C with

To perform paraformaldehyde fixation procedure, fixation solution is warmed in 37°C

permeable to antibodies. Therefore, cells do not need to be permeabilized.

disperse the tick detergent.

Table 4 and buffer pH is adjusted to 6,9 with NaOH.

Table 4. 2X PHEM Buffer Ingredients.

**4.2.1 Paraformaldehyde fixation**

stirring it for overnight.

water bath.

18,14 g Pipes 5,96 g Hepes 3,8 g EGTA 0,41 g MgCl2 up to 500 ml ddH2O

is a commonly used serum that is rich in mitogenic factors and preferred for proliferating cells; while neurons additionally require B – 27® supplement which is optimized serum substitute to support neurons in the culture.

As there are some disadvantages when serum is used to supplement culture medium, depending on the experimental conditions, plating medium can be changed with serum free medium, "Serum – Free Plating Medium" (Table 3). Since serum represents a major potential route for the introduction of agents including bacteria, fungi and viruses into cell culture, and serum containing media may be disruptive for the maintenance of the culture for a long period of time.

Table 1. Commonly used dissection medium for neurons.

Table 2. Commonly used plating medium for neurons.

Table 3. Commonly used serum – free plating medium for neurons.

#### **4.2 Cell fixation and permeabilization**

Cell fixation and permeabilization step not only provides access of the antibody to its antigen but also helps to preserve cells for longer storage periods. There are several methods for fixation and permeabilization of the cells. Choosing specific method depends on the specificity of the epitope and the antibodies. Aldehydes and organic solvents can be used for fixation of the cells to the slide. Aldehydes fix cells by denaturation and chemical modification of proteins. For instance, covalent reactions can occur with free amino groups of lysine residues and once protein cross linking occurs, cells become rigid. In aldehydes based fixation methods, cell membranes of intact cells remain relatively impenetrable to larger molecules such as antibodies; thus, "cell permeabilization" is required before immunostaining.

is a commonly used serum that is rich in mitogenic factors and preferred for proliferating cells; while neurons additionally require B – 27® supplement which is optimized serum

As there are some disadvantages when serum is used to supplement culture medium, depending on the experimental conditions, plating medium can be changed with serum free medium, "Serum – Free Plating Medium" (Table 3). Since serum represents a major potential route for the introduction of agents including bacteria, fungi and viruses into cell culture, and serum containing media may be disruptive for the maintenance of the culture

substitute to support neurons in the culture.

1X HBSS 10 mM HEPES

Table 1. Commonly used dissection medium for neurons.

5% FBS

Medium

Medium Table 3. Commonly used serum – free plating medium for neurons.

Table 2. Commonly used plating medium for neurons.

**4.2 Cell fixation and permeabilization**

immunostaining.

(100 U/mL – 100 g/mL) Penicillin – Streptomycin up to volume – ddH2O

up to volume – Neurobasal

up to volume – Neurobasal

Cell fixation and permeabilization step not only provides access of the antibody to its antigen but also helps to preserve cells for longer storage periods. There are several methods for fixation and permeabilization of the cells. Choosing specific method depends on the specificity of the epitope and the antibodies. Aldehydes and organic solvents can be used for fixation of the cells to the slide. Aldehydes fix cells by denaturation and chemical modification of proteins. For instance, covalent reactions can occur with free amino groups of lysine residues and once protein cross linking occurs, cells become rigid. In aldehydes based fixation methods, cell membranes of intact cells remain relatively impenetrable to larger molecules such as antibodies; thus, "cell permeabilization" is required before

0,3% D – Glucose 2% B – 27® Supplement 1 mM L – Glutamine

0,3% D – Glucose 2% B – 27® Supplement 1 mM L – Glutamine

for a long period of time.

Organic solvents such as methanol, ethanol, and acetone denature proteins without any covalent modifications. Their working principle is by removing bound H2O molecules of the cells. These solvents also remove membrane and some structural lipids, and cells become permeable to antibodies. Therefore, cells do not need to be permeabilized.

For immunostaining of cytoskeletal proteins, the most sensitive and effective fixation method is with aldehydes; glutaraldehyde and paraformaldehyde. Since aldehydes fixation method requires additional permeabilization step, a detergent, Triton – X100 can be used for cell extraction that leads cell permeabilization. Triton X100 is a detergent that is mostly used to extract the membrane from cells so that the cytoskeleton can be accessed. It is usually prepared as 10% solution in ddH2O and is rotated overnight in the cold room to fully disperse the tick detergent.

Another buffer called "PHEM (**P**ipes – **H**epes – **E**GTA – **M**gCl2) is also necessary for microtubule stabilization. Stock solution (2X PHEM) can be prepared as indicated in the Table 4 and buffer pH is adjusted to 6,9 with NaOH.

Table 4. 2X PHEM Buffer Ingredients.

Cell permeabilization and fixation can either be performed separately or together in one step as termed fixation/co – extraction depending on the design of the study. For instance, in our p60 katanin – microtubule co – localization studies we preferred simultaneous fixation/co – extraction method. Since p60 katanin severs microtubules into short pieces, fixing the cells first would clutter inner side of the cells with short pieces/subunits of tubulin and therefore would cause to have higher background with the antibody staining. Whereas, if the fixation/co – extraction steps were performed simultaneously, it would allow some of the tubulin subunits to be extracted from the cell and therefore would give a cleaner staining while keeping some of the short microtubule pieces inside the cells. On the other hand, if the extraction step were to be done at first, as it would cause most of the short pieces to leave the cell, it may not be possible to see short microtubule pieces that are created by katanin. Therefore, as in the case of the examples, one should decide about the methodology of the cell permeabilization and fixation depending on the need of the question asked.

Below you can see some examples for different cell fixation protocols commonly used in the fixation of microtubules and microtubule related proteins in dividing cells.

#### **4.2.1 Paraformaldehyde fixation**

Immunocytochemistry of Cytoskeleton Proteins 113

 Cells are quenched 2 times for 15 minutes with 2 mg/ml sodium borohydrate solution. Following final rinse with PBS 3 times for 5 minutes, cells can be kept at 4°C until the

0,2%Glutaraldehyde

up to final volume – ddH2O

Fig. 16. Immunostaining of p60 katanin in glutaraldehyde fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red) and Alexa Fluor 488 goat anti – mouse IgG (green).

Since organic solvents also permeabilize cell membrane, additional protein extraction step is not required in methanol fixation method. Addition of nearly 200 μl of ice cold methanol per slide and placing at – 20°C for 10 minutes is enough to obtain fixation of the cells. Cells can

Exact and sharp location identification of p60 katanin protein is crucial for some critical places such as on mitotic spindle and on branch points of axons and dendrites. Since good protein visualization is obtained upon optimum fixation method and optimum fixation method changes depending on the epitope and antibody specificity, we have tested our home-made 1G6 antibody for p60 katanin under different fixation methods including paraformaldehyde fixation (Fig.15), glutaraldehyde fixation (Fig.16), and methanol fixation (Fig.17). 1G6 gave the best results with paraformaldehyde, whereas other fixation methods

10µM Taxol 1X PHEM

Table 6. Fixative ingredients for glutaraldehyde fixation.

Images were obtained with laser scanning confocal microscope.

caused fuzzy appearance of the p60 katanin protein.

be rinsed with PBS 3 times for 5 minutes and are ready for blocking step.

**4.2.3 Methanol fixation**

Cells are rinsed with PBS 3 times for 5 minutes.

day of immunostaining.

Table 5. Fixative ingredients for paraformaldehyde fixation.

Fig. 15. Immunostaining of p60 katanin in paraformaldehyde fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red). Images were obtained with laser scanning confocal microscope.

#### **4.2.2 Glutaraldehyde fixation**

Petri dishes containing the cultured cells are filled with fixation solution and incubated

 Free aldehyde groups which are formed during the fixation procedure may decrease antigenic site accessibility. To prevent this, Sodium Borohydrate treatment has to be performed. 2 mg/ ml sodium borohydrate solution is prepared in PBS. Cells are

quenched 2 times for 15 minutes with 2 mg/ ml sodium borohydrate solution. Following final rinse with PBS 3 times for 5 minutes, cells can be kept at 4°C until the

Chemical Final Concentration

ddH2O up to final volume

Fig. 15. Immunostaining of p60 katanin in paraformaldehyde fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red). Images were obtained with laser scanning confocal

Petri dishes containing the cultured cells are filled with fixation solution and incubated

Following fixation, permeabilization step can be performed by keeping cells in 0,1%

The glutaraldehyde fixation solution is warmed in 37°C water bath.

Fixed cells are rinsed with 1XPBS 3 times for 5 minutes.

Triton X100 for 10 minutes at room temperature.

Paraformaldehyde 4% PHEM 1X

Table 5. Fixative ingredients for paraformaldehyde fixation.

 Fixed cells are rinsed with 1X Phosphate Buffered Saline (PBS) 3 times for 5 minutes. Following fixation; permeabilization step can be performed by keeping cells in 0,1%

at room temperature for 15 minutes.

day of immunostaining.

microscope.

**4.2.2 Glutaraldehyde fixation**

at room temperature for 15 minutes.

Triton X100 for 10 minutes at room temperature. Cells are rinsed with PBS 3 times for 5 minutes.

Table 6. Fixative ingredients for glutaraldehyde fixation.

Fig. 16. Immunostaining of p60 katanin in glutaraldehyde fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red) and Alexa Fluor 488 goat anti – mouse IgG (green). Images were obtained with laser scanning confocal microscope.

#### **4.2.3 Methanol fixation**

Since organic solvents also permeabilize cell membrane, additional protein extraction step is not required in methanol fixation method. Addition of nearly 200 μl of ice cold methanol per slide and placing at – 20°C for 10 minutes is enough to obtain fixation of the cells. Cells can be rinsed with PBS 3 times for 5 minutes and are ready for blocking step.

Exact and sharp location identification of p60 katanin protein is crucial for some critical places such as on mitotic spindle and on branch points of axons and dendrites. Since good protein visualization is obtained upon optimum fixation method and optimum fixation method changes depending on the epitope and antibody specificity, we have tested our home-made 1G6 antibody for p60 katanin under different fixation methods including paraformaldehyde fixation (Fig.15), glutaraldehyde fixation (Fig.16), and methanol fixation (Fig.17). 1G6 gave the best results with paraformaldehyde, whereas other fixation methods caused fuzzy appearance of the p60 katanin protein.

Immunocytochemistry of Cytoskeleton Proteins 115

 Primary antibody containing cells are incubated at 4°C for overnight. During this staining period, the cover glasses are placed in a "humidified chamber," in a large Petri dish containing moistened filter paper, which minimizes evaporation of the antibody. Primary antibody is removed by vacuum carefully and dishes are washed by using PBS

Cells are then blocked again for 1 hour at room temperature with appropriate blocking

Finally 6 – 7 drops of anti – fade mounting medium (Table 7) is added on the slides in

Immunocytochemistry is a powerful biochemical technique to identify the inner worlds of cells. Capturing the intracellular architecture and dynamics of cells as close as to their physiological state would require visualization and image analysis with state of the art techniques upon immunostaining with highly specific monoclonal antibodies and optimized

Therefore, there should be much effort to obtain highly specific monoclonal antibodies for a wide range of applications, and antibodies on the market would require much more

We would like to thank all the past and present members of Karabay Molecular

Akkor, M., Karabay, A. (2010). Production and characterization of a monoclonal antibody

Errico, A., Claudiani, P., D'Addio, M., Rugarli, E.I. (2004). Spastin interacts with the

the distal axon. *Human Molecular Genetics*, 13, 182121–182132, 0964 – 6906

centrosomal protein NA14, and is enriched in the spindle pole, the midbody and

vigorous characterization steps before they are made commercially available.

Neurobiology Lab for their contribution to all the work presented here.

against P60 – katanin, *Hybridoma,* 6:531 – 7, 1554 – 0014

 Cover slips are placed on the cells and fixed from the edges by using nail polisher. Cells are visualized and analyzed by using fluorescent or confocal microscopy.

0,106 gr N – Propyl gallate

 Secondary antibody is diluted and centrifuged in the same way as primary antibody. Diluted secondary antibody solution is put on the slide and cells are incubated for 1 hour in 37°C incubator. Secondary antibody application requires working in dark.

Diluted primary antibody solution is put on the slide containing fixed cells.

3 times for 5 minutes each.

Cells are washed with PBS 3 times for 5 minutes each.

order to prevent rapid fading of fluorescent signals.

5 ml PBS 45 ml glycerol

Table 7. Ingredients for mounting medium.

solution.

**5. Conclusion**

**6. Acknowledgement**

**7. References**

fixation.

Fig. 17. Immunostaining of p60 katanin in methanol fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red) and Alexa Fluor 488 goat anti – mouse IgG (green). Images were obtained with laser scanning confocal microscope.

#### **4.3 Blocking for immunostaining**

Before the antibody application part of the immunostaining procedure, cells should be blocked with an appropriate blocking solution to reduce nonspecific bindings of antibodies. To prepare blocking solution, 10% appropriate serum (goat or donkey depending on the type of secondary antibody) and 10 mg/ml Bovine Serum Albumin (BSA) is dissolved in PBS. Cells are then incubated in this blocking solution at room temperature for 1 hour or any optimized time period.

#### **4.4 Immunostaining**

Antibodies are the main players of immunostaining procedure. Antibodies either can be applied in a single step or in two steps. In single – step method, antibodies are directly conjugated to a colored agent, fluorochrome; whereas in two – step method the first antibody does not have the chemical structure to support a colored agent. In this case, following the application of the primary (colorless) antibody, a secondary antibody linked to a colored agent is applied, where the secondary antibody binds to the primary. The colored agent can then be visualized under the microscope. Comparing to the single – step method, two – step method has some advantages. Variety of coloring agents can be conjugated to any given type of secondary antibody which gives chance to visualize different types of proteins at the same time.

Antibodies are diluted to obtain working stock depending on the manufacturer's instructions, but they usually require optimization studies to find the exact dilution ratio for the antibody of interest. To obtain clear visualization, any precipitates which may be formed during storage need to be cleaned by centrifuging the diluted antibodies at 10.000 rpm for 10 minutes at 4°C before use. Below you can find a sample for two – step immunostaining procedure.

Fig. 17. Immunostaining of p60 katanin in methanol fixed HeLa cells. p60 katanin was labeled with 1G6 mouse anti – p60 katanin primary antibody and visualized with Alexa Fluor 546 goat anti – mouse IgG (red) and Alexa Fluor 488 goat anti – mouse IgG (green).

Before the antibody application part of the immunostaining procedure, cells should be blocked with an appropriate blocking solution to reduce nonspecific bindings of antibodies. To prepare blocking solution, 10% appropriate serum (goat or donkey depending on the type of secondary antibody) and 10 mg/ml Bovine Serum Albumin (BSA) is dissolved in PBS. Cells are then incubated in this blocking solution at room temperature for 1 hour or

Antibodies are the main players of immunostaining procedure. Antibodies either can be applied in a single step or in two steps. In single – step method, antibodies are directly conjugated to a colored agent, fluorochrome; whereas in two – step method the first antibody does not have the chemical structure to support a colored agent. In this case, following the application of the primary (colorless) antibody, a secondary antibody linked to a colored agent is applied, where the secondary antibody binds to the primary. The colored agent can then be visualized under the microscope. Comparing to the single – step method, two – step method has some advantages. Variety of coloring agents can be conjugated to any given type of secondary antibody which gives chance to visualize different types of

Antibodies are diluted to obtain working stock depending on the manufacturer's instructions, but they usually require optimization studies to find the exact dilution ratio for the antibody of interest. To obtain clear visualization, any precipitates which may be formed during storage need to be cleaned by centrifuging the diluted antibodies at 10.000 rpm for 10 minutes at 4°C

before use. Below you can find a sample for two – step immunostaining procedure.

Images were obtained with laser scanning confocal microscope.

**4.3 Blocking for immunostaining**

any optimized time period.

proteins at the same time.

**4.4 Immunostaining**

Table 7. Ingredients for mounting medium.

#### **5. Conclusion**

Immunocytochemistry is a powerful biochemical technique to identify the inner worlds of cells. Capturing the intracellular architecture and dynamics of cells as close as to their physiological state would require visualization and image analysis with state of the art techniques upon immunostaining with highly specific monoclonal antibodies and optimized fixation.

Therefore, there should be much effort to obtain highly specific monoclonal antibodies for a wide range of applications, and antibodies on the market would require much more vigorous characterization steps before they are made commercially available.

#### **6. Acknowledgement**

We would like to thank all the past and present members of Karabay Molecular Neurobiology Lab for their contribution to all the work presented here.

#### **7. References**

**6**

**-Dependent**

*Okayama University*

*Japan*

**Immunocytochemical Approaches**

Chiharu Sogawa, Norio Sogawa and Shigeo Kitayama

**/Cl-**

**Neurotransmitter Transporters**

**to the Identification of Membrane Topology**

Transporters, located in the cell surface plasma membrane and intracellular organelle membrane, compose a major class of integral membrane proteins, as distinguished with their specialized functions as channels and receptors. They are divided into two groups of gene family; one is the solute carrier (SLC) super family and the other is the ATP-binding cassette (ABC) gene family (Giacomini & Sugiyama, 2006). The transporters for neurotransmitters in the plasma membrane, which belong to the SLC6 gene family,

neurotransmitters into the neuronal and/or glial cells (Iversen, 1971). They are responsible for the reuptake not only of neurotransmitters, monoamines (dopamine (DA), noradrenaline (NA), and serotonin (5-HT)) and amino acids (-aminobutyric acid (GABA) and glycine), but also of neuromodulators and/or osmolytes (proline, taurine, betaine, and creatine). Cloning of their cDNA has facilitated the understanding of the primary structure, gene expression, and their roles in neuronal functions (Amara & Kuhar, 1993). Cellular and molecular aspects of these transporters are needed for the clarification of their physiological and pathological relevance, since it seems likely that alterations of their structure, function and expression produce their anatomical and functional divergence, resulting in an involvement of a

Elucidating the molecular structure of these transporters should be a first step to clarify their functional mechanisms, such as transport processes, regulations and consequent phenomena. Particularly, information of the membrane topology is substantially important

neurotransmitter transporters, this chapter focuses on the monoamine neurotransmitter transporters such as those for DA (DAT), NA (NET), and 5-HT (SERT). These monoamine transporters are of particular interest, since they are a target of drugs of abuse and/or antidepressants, and are involved in various neuronal disorders (Gether et al., 2006). Therefore, understanding of the expression and function of these transporters could promise clues to clarify their pathophysiological significance and thereby to develop medications for

for clarifying their structure-function relationship. Among the Na+/Cl-

terminate synaptic neurotransmission by Na+/Cl-

number of neurological and psychiatric disorders.

treatments of neuronal disorders mentioned above.

**1. Introduction**

**of the Na+**

## **Immunocytochemical Approaches to the Identification of Membrane Topology of the Na+ /Cl- -Dependent Neurotransmitter Transporters**

Chiharu Sogawa, Norio Sogawa and Shigeo Kitayama *Okayama University Japan*

### **1. Introduction**

116 Applications of Immunocytochemistry

Karabay, A., Yu, W., Solowska, J. M., Baird, D. H., Baas, P. W. (2004). Axonal growth is

Lodish, H. & Baltimore, D., (1995). *Molecular Cell Biology* (3rd edition). Scientific American

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Transporters, located in the cell surface plasma membrane and intracellular organelle membrane, compose a major class of integral membrane proteins, as distinguished with their specialized functions as channels and receptors. They are divided into two groups of gene family; one is the solute carrier (SLC) super family and the other is the ATP-binding cassette (ABC) gene family (Giacomini & Sugiyama, 2006). The transporters for neurotransmitters in the plasma membrane, which belong to the SLC6 gene family, terminate synaptic neurotransmission by Na+/Cl--dependent uptake of released neurotransmitters into the neuronal and/or glial cells (Iversen, 1971). They are responsible for the reuptake not only of neurotransmitters, monoamines (dopamine (DA), noradrenaline (NA), and serotonin (5-HT)) and amino acids (-aminobutyric acid (GABA) and glycine), but also of neuromodulators and/or osmolytes (proline, taurine, betaine, and creatine). Cloning of their cDNA has facilitated the understanding of the primary structure, gene expression, and their roles in neuronal functions (Amara & Kuhar, 1993). Cellular and molecular aspects of these transporters are needed for the clarification of their physiological and pathological relevance, since it seems likely that alterations of their structure, function and expression produce their anatomical and functional divergence, resulting in an involvement of a number of neurological and psychiatric disorders.

Elucidating the molecular structure of these transporters should be a first step to clarify their functional mechanisms, such as transport processes, regulations and consequent phenomena. Particularly, information of the membrane topology is substantially important for clarifying their structure-function relationship. Among the Na+/Cl- -dependent neurotransmitter transporters, this chapter focuses on the monoamine neurotransmitter transporters such as those for DA (DAT), NA (NET), and 5-HT (SERT). These monoamine transporters are of particular interest, since they are a target of drugs of abuse and/or antidepressants, and are involved in various neuronal disorders (Gether et al., 2006). Therefore, understanding of the expression and function of these transporters could promise clues to clarify their pathophysiological significance and thereby to develop medications for treatments of neuronal disorders mentioned above.

Immunocytochemical Approaches to the Identification

fixation and permeabilization of cell cultures.

**A**

**B**

5 4 2

of Membrane Topology of the Na+/Cl--Dependent Neurotransmitter Transporters 119

comparison of their benefits and problems. In addition, we summarized basic methods concerning cell preparations, gene transfection techniques into mammalian cell lines, and

Fig. 1. Schematic presentation of the topological model of the neurotransmitter transporter (A) and LeuT (B). (A) Transmembrane helices are shown as cylinders and are numbered 1- 12. Helical features are based on the hydropathy analysis of deduced amino acid sequence of the cloned transporter cDNA. The C and N termini are intracellular. (B) Model of LeuT in the plasma membrane based on its crystal structure. S: substrate leucine, closed circle: Na+.

**NH2**

1a 6b

1b 6a S

**COOH NH2**

1 2 3 4 5 6 7 8 9 10 11 12

EL4

7 9 10 11 12

**in**

**out**

**COOH**

[out]

[in]

Cloning of their cDNA has facilitated the understanding of the primary structure, gene expression, and their roles in neuronal functions. The GABA transporter-1 (GAT-1) cDNA was cloned from rat brain as a first one of the gene family by means of protein purification (Guastella et al., 1990). Hydropathy analysis of the deduced protein suggested multiple TMDs that did not display homology to any previously identified proteins as assessed by database search, indicating that GAT-1 appeared to be a member of a previously

Soon after the cloning of GAT-1, human NET cDNA was cloned by direct expression of pools of clones from a human SK-N-SH cell cDNA library in COS-1 cells and screening for

**2. General consideration of the neurotransmitter transporter**

**2.1 Cloning of cDNAs**

uncharacterized family of transporter.

Cloning of the GABA transporter (GAT) cDNA and subsequently the NET cDNA have led to the identification of the gene family, such as SLC6 (Amara & Kuhar, 1993). Hydropathy analysis1 of the primary structure of these transporters showed a common model for their membrane topology; the twelve hydrophobic regions that consist of -helical transmembrane domains (TMDs) interrupted by alternating intra- and extracellular loops, one large putatively extracellular loop, and intracellularly localization of N- and C-termini (Fig. 1A). Based on this information, initial studies have examined the membrane topology of the transporters using various biochemical techniques including, for example, an immunological technique with specific antibodies against hydrophilic regions predicted as intra- and extracellular loops, or antibodies against known epitope tags incorporated at hydrophilic regions of the neurotransmitter transporters in the transiently or stably expressing cell lines. Recent success of the X-ray crystallography of the bacterial homologue of the Na+/Cl- -dependent neurotransmitter transporter LeuT confirmed the proposed model and extended the structural understanding (Yamashita et al., 2005) (Fig. 1B). However, it is still difficult to understand fully the structure of mammalian plasma membrane neurotransmitter transporters. In addition to more studies on X-ray crystallography of bacterial homologues, we are needed to address directly to the mammalian transporter proteins to determine their membrane topology.

We have been investigating the expression and function of DAT and NET isoforms produced by alternative RNA splicing (Kitayama & Dohi, 2003). Recently, we found novel variants of human DAT (hDAT) and NET, skipping the region encoded by exon 6 (Sogawa et al., 2010). A hydropathy analysis of the variant designated hDATEX6 revealed 11 putative TMDs, suggesting a membrane topology different from that of full-length (FL) hDAT. To explore the unique structure of this variant, an immunocytochemical analysis with confocal microscopy was performed using two specific antibodies, one recognizing the second extracellular region (anti-hDAT-EL2 antibody) and the other recognizing the intracellular C-terminus (anti-hDAT-Ct antibody) in the tranfected cells treated with (permeabilized) or without (non-permeabilized) surfactant Triton. Immunoreactivity to the anti-hDAT-Ct antibody was only observed in FL hDAT-expressing cells treated with Triton, in contrast to the detection even in the untreated cells expressing hDATEX6, strongly suggesting the C-terminus of hDATEX6 to be located extracellulary. This information of the membrane topology is substantially important for clarifying the structure-function relationship. Since changes in membrane topology could influence the expression and function of the transporter at large, our observations provide clue to understand the functional modifications and expressional alterations of the DAT/NET splice variants.

In this chapter, we summarize the recent progress in our understanding of the contribution of the immunocytochemical approaches to determining a membrane topology of the plasma membrane neurotransmitter transporters. We discuss a usefulness of specific antibodies against the epitopes located in the extra- and/or intra-cellular region of plasma membrane protein, that led us to identify the unique structure of the DAT/NET splice variants. We also discuss the additional molecular biological and protein engineering techniques in

<sup>1</sup> An index that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence, thereby predicts higher structure of proteins. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 10-20 amino acid side-chains is taken into consideration.

comparison of their benefits and problems. In addition, we summarized basic methods concerning cell preparations, gene transfection techniques into mammalian cell lines, and fixation and permeabilization of cell cultures.

Fig. 1. Schematic presentation of the topological model of the neurotransmitter transporter (A) and LeuT (B). (A) Transmembrane helices are shown as cylinders and are numbered 1- 12. Helical features are based on the hydropathy analysis of deduced amino acid sequence of the cloned transporter cDNA. The C and N termini are intracellular. (B) Model of LeuT in the plasma membrane based on its crystal structure. S: substrate leucine, closed circle: Na+.

## **2. General consideration of the neurotransmitter transporter**

## **2.1 Cloning of cDNAs**

118 Applications of Immunocytochemistry

Cloning of the GABA transporter (GAT) cDNA and subsequently the NET cDNA have led to the identification of the gene family, such as SLC6 (Amara & Kuhar, 1993). Hydropathy analysis1 of the primary structure of these transporters showed a common model for their membrane topology; the twelve hydrophobic regions that consist of -helical transmembrane domains (TMDs) interrupted by alternating intra- and extracellular loops, one large putatively extracellular loop, and intracellularly localization of N- and C-termini (Fig. 1A). Based on this information, initial studies have examined the membrane topology of the transporters using various biochemical techniques including, for example, an immunological technique with specific antibodies against hydrophilic regions predicted as intra- and extracellular loops, or antibodies against known epitope tags incorporated at hydrophilic regions of the neurotransmitter transporters in the transiently or stably expressing cell lines. Recent success of the X-ray crystallography of the bacterial homologue

and extended the structural understanding (Yamashita et al., 2005) (Fig. 1B). However, it is still difficult to understand fully the structure of mammalian plasma membrane neurotransmitter transporters. In addition to more studies on X-ray crystallography of bacterial homologues, we are needed to address directly to the mammalian transporter

We have been investigating the expression and function of DAT and NET isoforms produced by alternative RNA splicing (Kitayama & Dohi, 2003). Recently, we found novel variants of human DAT (hDAT) and NET, skipping the region encoded by exon 6 (Sogawa et al., 2010). A hydropathy analysis of the variant designated hDATEX6 revealed 11 putative TMDs, suggesting a membrane topology different from that of full-length (FL) hDAT. To explore the unique structure of this variant, an immunocytochemical analysis with confocal microscopy was performed using two specific antibodies, one recognizing the second extracellular region (anti-hDAT-EL2 antibody) and the other recognizing the intracellular C-terminus (anti-hDAT-Ct antibody) in the tranfected cells treated with (permeabilized) or without (non-permeabilized) surfactant Triton. Immunoreactivity to the anti-hDAT-Ct antibody was only observed in FL hDAT-expressing cells treated with Triton, in contrast to the detection even in the untreated cells expressing hDATEX6, strongly suggesting the C-terminus of hDATEX6 to be located extracellulary. This information of the membrane topology is substantially important for clarifying the structure-function relationship. Since changes in membrane topology could influence the expression and function of the transporter at large, our observations provide clue to understand the functional modifications and expressional alterations of the DAT/NET splice variants.

In this chapter, we summarize the recent progress in our understanding of the contribution of the immunocytochemical approaches to determining a membrane topology of the plasma membrane neurotransmitter transporters. We discuss a usefulness of specific antibodies against the epitopes located in the extra- and/or intra-cellular region of plasma membrane protein, that led us to identify the unique structure of the DAT/NET splice variants. We also discuss the additional molecular biological and protein engineering techniques in

1 An index that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence, thereby predicts higher structure of proteins. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 10-20 amino

of the Na+/Cl-

acid side-chains is taken into consideration.

proteins to determine their membrane topology.

Cloning of their cDNA has facilitated the understanding of the primary structure, gene expression, and their roles in neuronal functions. The GABA transporter-1 (GAT-1) cDNA was cloned from rat brain as a first one of the gene family by means of protein purification (Guastella et al., 1990). Hydropathy analysis of the deduced protein suggested multiple TMDs that did not display homology to any previously identified proteins as assessed by database search, indicating that GAT-1 appeared to be a member of a previously uncharacterized family of transporter.

Soon after the cloning of GAT-1, human NET cDNA was cloned by direct expression of pools of clones from a human SK-N-SH cell cDNA library in COS-1 cells and screening for

Immunocytochemical Approaches to the Identification

impermeant reagents (Fig. 2D)

unaltered (Javitch, 1998).

**3.1 Site-directed antibodies**

of Membrane Topology of the Na+/Cl--Dependent Neurotransmitter Transporters 121

3. Determination of the orientation of a reporter domain linked to a series of C-terminal

4. Chemical modification of specific amino acid residues using membrane-permeant and -

5. Immunocytochemical detection of the epitope-tag inserted into hydrophilic domains or

Each approach has its own benefit but also limitation, because many of the approaches require modification of the protein being studied, which often results in changes of the functional properties due to perturbation of the structure of the protein (Green et al., 2001). The neurotransmitter transporter protein is not tolerant of the alterations in primary sequence at conserved domain, such as the insertion of reporter epitope or the mutagenesis of several amino acid residues. Therefore, it appears to allow minor modification, such as substitution of single amino acid residue with cysteine, for example. Since substitution with cysteine appears to be very tolerated, it is useful for evaluating the membrane topology to determine the intra- and extracellular orientation of substituted cysteine residues using membrane-permeant and -impermeant sulfhydryl-reactive agents. A residue which is water-accessible from the cytoplasmic side but not from the extracellular side of the membrane should not react at an appreciate rate with extracellularly applied MTSET or MTSES (impermeant reagent), but might react with MTSEA (permeant reagent). In this way, topology can be assessed under conditions in which protein function and structure remain

All members of the Na+/Cl—dependent neurotransmitter transporter gene family share the twelve hydrophobic regions that might consist of α-helical transmembrane domains (TMDs) interrupted by alternating intra- and extracellular loops, one large putatively extracellular loop, and intracellularly localization of N- and C-termini. Based on this information, initial studies examined the membrane topology of the transporters by various biochemical techniques. For example, study with immunological technique using specific antibodies against hydrophilic regions predicted as intra- and extracellular loops or antibodies against known epitope tags incorporated at hydrophilic regions of the neurotransmitter transporters transiently or stably expressed in the cell lines, strongly

Mabjeesh and Kanner have reported the first evidence for the membrane topology of GAT using antibodies raised against synthetic peptides corresponding to several regions of the rat brain GAT-1 (Mabjeesh & Kanner, 1992). According to the model based on the hydropathy analysis, these 4 antibodies against amino and carboxy termini and predicted 3rd and 4th intracellular loops recognized the intact transporter on Western blots. Interestingly, GAT protein digested partially at amino and carboxy termini by protease revealed transport activity when reconstituted in liposomes, suggesting that these regions are not essential for transport function (Mabjeesh and Kanner, 1992). These studies did not address directly to explore the membrane topology of the transporter, however, provided