Rab24 is localized to perinuclear reticular structures that partially colocalize with marker proteins for ER, cis-Golgi, and ER-Golgi intermediate compartments [46]. Under starvation conditions, Rab24 relocated to large vesicles, where LC3 was localized. The appearance of these vesicles was enhanced in the presence of vinblastin, an agent that disrupts microtubules and prevents fusion of autophagosomes with lysosomes [46]. Interestingly, since no such distri‐ bution change was observed in cells expressing the mutant Rab24S67L that introduced the mutation into the GTP-binding motif, normally functioning Rab24 protein appears to be required for the formation of autophagosomes in response to starvation. *Coxiella burnetii* survives and replicates in MDC- and LC3-positive phagolysosomal compartments and Rab7 participates in the formation of *Coxiella*-containing vacuoles [26]. Overexpression of Rab24 or LC3 also accelerated the occurrence of *Coxiella*-containing vacuoles early after infection [47]. The expression of the Rab24 mutant (Rab24S67L), which does not localize to autophagosomes, significantly reduced the number and size of the phagolysosomal structures, although the inhibitory effect was not enduring but mutant expression delayed the generation of phagoly‐ sosomes [47]. Taken together, these results suggest that overexpression of proteins involved in the autophagic pathway, such as Rab24, increases the development of phagolysosomes for

Rab24 is also supposed to contribute to the degradation of aggregated proteins in rat cardiac myocytes. Glucose deprivation induced the formation of aggregates and aggresomes of polyubiquitinated proteins, and then they colocalized with exogenously expressing green

induced autophagy.

*Coxiella* replication.

**5. Rab24 GTPase and autophagy**

52 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Mouse Rab32 and Rab38 operate in a functionally redundant manner in regulating skin melanocyte pigmentation and regulate post-Golgi trafficking of tyrosinase and tyrosinaserelated protein 1, thereby suggesting their critical roles in melanosome maturation [49]. In *Xenopus* melanophores, Rab32 is involved in the regulation of melanosome transport by cAMPdependent protein kinase A [50]. Although Rab32 is expressed in most human tissues [51], little is known about the physiological roles of Rab32 in tissues and cells other than melanocytes.

#### **6.1. Rab32 GTPase and constitutive autophagy**

Rab32 is supposed to localize to ER [52] and ER and mitochondria [53]. We showed previously that Rab32 participates in constitutive autophagy in HeLa cells derived from cervical cancer [52]. The expressed wild-type or GTP-bound active form of human Rab32 was primarily localized to the ER. Interestingly, overexpression of the wild-type or active form of Rab32 induced the formation of autophagic vacuoles containing LC3, the ER-resident protein calnexin and late endosomal/lysosomal membrane protein LAMP-2 even under nutrient-rich conditions. Moreover, the localization of Rab32 to ER was necessary for the formation of autophagosomes [52], because the expression of a mutant Rab32 deleted two cysteine residues that are essential for association with the membrane, impaired autophagy vacuole formation [50]. There is a long-standing debate concerning from where the autophagosomal membrane is derived. So far, two possibilities have been proposed: it arises from pre-existing organelles, such as the ER or Golgi, or from de novo formation [54]. Our findings mentioned above postulate, therefore, that Rab32 facilitates the formation of autophagic vacuoles whose membranes are derived from the ER. In addition, expression of the inactive form of Rab32 or depletion of Rab32 expression by siRNA caused the formation of p62 and ubiquitinated protein-accumulating aggresomes and prevented constitutive autophagy [52]. Thus, these results imply the physiological importance of Rab32 in the cellular clearance of aggregated proteins by basal constitutive autophagy.

As well as Rab7, Rab32 also seems to participate in phagosome maturation in pathogeninduced autophagic degradation by infection with *Salmonella enterica serovar Typhimurium* [55] or *Mycobacterium tuberculosis*[56], especially in the recruitment of lysosomal enzyme cathepsin D to phagosomes containing *M. tuberculosis* [56]. Rab32 and some other Rabs localized to *M. tuberculosis*-containing phagosomes transiently, and the expression of the inactive form of Rab32 showed the impairment of its recruitment to phagosomes [56], but had no effect on phagosomal fusion with lysosomes [55]. Therefore, these results imply that Rab32 regulates the recruitment of cathepsin D to the phagosomes.

neuroendocrine cells, not only in the Golgi [68]. It has been shown that both Rab33A and Rab33B interact with Atg16L, which associates with Atg5-12 complex on an isolation mem‐ brane for the duration of autophagosome formation and is essential for canonical autophagy [69]. These interactions occur in a GTP-dependent manner [70]. Expression of the active form of Rab33B induced the lipidation of LC3 even under nutrient-rich conditions, but this was not caused by the blockage of autophagic degradation [70]. Furthermore, this form of Rab33B inhibited constitutive autophagy, but not starvation-induced autophagy, as judged by the degradation of p62/SQSTM1, a substrate of autophagy [70]. The precise reason for this dysfunction is unknown, but it is possible to speculate that the active form of Rab33B recruits Atg5-12/16L complex to incorrect membranes because no obvious large LC3 dots were observed in cells expressing this mutant. Rab33B has another interaction partner, OATL1, a RabGAP, which can bind LC3 [71]. Rab33B seems to be a target substrate of OATL1 and to be involved in the fusion between autophagosomes and lysosomes. Overexpression of the wildtype or active form of Rab33B inhibits the fusion between autophagosomes and lysosomes, because OATL1 inactivates Rab33B as a GAP protein, and this inhibition leads to the blockade of autophagic flux. In fact, the expression of the active form of Rab33B increased the amount of LC3-II as previously reported [70], but did not show the colocalization of lysosomal membrane protein LAMP-1 with LC3-positive dots [71], suggesting that autophagosomes in the active form of Rab33B-expressing cells cannot efficiently fuse with lysosomes. Very recently, it was revealed that Atg16L interacts with Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L in neuroendocrine PC12 cells [72]. Knockdown of endogenous Atg16L in PC12 cells caused marked inhibition of hormone secretion inde‐ pendently of autophagic activity [72], indicating that Atg16L controls autophagy in all cell types as well as secretion from dense-core vesicles, presumably by acting as a Rab33A effector,

Rab GTPases in Autophagy http://dx.doi.org/10.5772/54578 55

Based on the results of a large body of research, regulation of autophagy by Rab proteins could be loosely classified into two types, (i) autophagosome formation or (ii) autophagosome maturation (i.e. the fusion of autophagosomes with lysosomes) [Figure 2]. We assumed that Rab7, Rab9, and Rab11 could be sorted into type (ii) profile, and Rab24, Rab32 and Rab33 into type (i). Autophagy indicates not only the sequestration of materials into autophagosomes but also the degradation by lysosomal enzymes. Therefore, Rab proteins associated with the fusion step of autophagosomes with lysosomes have a fatal role in autophagic flux, and Rabs also trigger autophagosome formation. Type (ii)-categorized Rab proteins are mostly localized at late endosomes and play a role in membrane traffic to lysosomes, recycling endosomes or TGN. It is conceivable that they are involved in autophagosome maturation, because late endosomes, TGN, recycling endosomes, or Golgi-derived vesicles fuse with lysosomes routinely [73]. So far, no clear result has shown that Rab24 and Rab33 directly associate with the formation of autophagosomes except for Rab32, which is associated with ER and/or mitochondria and is regarded as a key factor for the supply to autophagosome formation [52]. However, it could

in neuroendocrine cells.

**8. Conclusion**

### **6.2. Rab32 GTPase and interaction molecules**

Recently, it has been reported that, in *Drosophila*, Rab32 colocalized with LysoTracker labeling lysosomes and GFP-Atg8 (LC3 homologue), indicating that Rab32 is localized at lysosomes and/or autophagosomes during programmed autophagy for metamorphosis to differentiate the fat body, salivary gland, and midgut [57]. Previously, Ma et al. reported that the Claret encoded by *claret*, a member of the granule group eye color genes [58], coprecipitated not only with Rab-RP1, a Rab GTPase encoded by *Drosophila lightoid*, but also with its human homo‐ logues, Rab32 and Rab38 [59]. Furthermore, the autophagosome formation was impaired in Rab32/*lightoid* mutants and Rab32 GEF/*claret* mutants, suggesting that Rab32 activity is required for the autophagic process of the fat body [57]. Previously, it has been suggested that autophagy impairment reduces lipid accumulation and impairs adipocytes differentiation in mice [60, 61]. In fact, downregulation of autophagy in *Drosophila* led to a decrease in the size of lipid droplets in *atg*-related genes in knocked down *Drosophila* fat body cells [57]; therefore, Rab32 appears to regulate lipid storage by controlling autophagy. Another report showed that Rab32 is upregulated in the epidermis and midgut during metamorphosis in *Helicoverpa armigera* [62], suggesting that Rab32 may participate in organeogenesis in insects.

In addition to GEF, a GAP for Rab32, RUTBC1, was identified [63]. RUTBC1 is a TBC domaincontaining protein that binds to Rab9A in a nucleotide binding-state-dependent manner both in vitro and in vivo but has no GAP activity for Rab9A [63]; however, RUTBC1 acts as a GAP for Rab32 and Rab33B, and its TBC domain stimulates GTP hydrolysis [63]. Therefore, RUTBC1 may function in the autophagy process, as both Rab32 and Rab33B are suggested to be regulatory factors of autophagy.

#### **6.3. Rab32 GTPase and disease**

Recently, *RAB32* and *IL23R (interleukin receptor 23)* were identified as susceptibility genes for leprosy in a genome-wide association study [64], although *IL23R* was previously reported to be a gene involved in Crohn's disease [65]. Leprosy, also known as Hansen's disease, is a chronic granulomatous infectious disease caused by *Mycobacterium leprae*, which affects both peripheral nerves and mucosa of the upper respiratory tract. As Rab32 participates in regu‐ lating the recruitment of cathepsin D to phagosomes containing *M. tuberculosis* [56], referred to above, Rab32 may have function in the pathogenesis of leprosy, such as host defense against *M. leprae* infection.

## **7. Rab33 GTPase and autophagy**

Rab33 has two isoforms, Rab33A and Rab33B. Rab33B is expressed ubiquitously, although Rab33A is expressed exclusively in the brain and cells of the immune system [66]. Rab33B is localized at the Golgi apparatus [67], although Rab33A also targets dense-core vesicles in neuroendocrine cells, not only in the Golgi [68]. It has been shown that both Rab33A and Rab33B interact with Atg16L, which associates with Atg5-12 complex on an isolation mem‐ brane for the duration of autophagosome formation and is essential for canonical autophagy [69]. These interactions occur in a GTP-dependent manner [70]. Expression of the active form of Rab33B induced the lipidation of LC3 even under nutrient-rich conditions, but this was not caused by the blockage of autophagic degradation [70]. Furthermore, this form of Rab33B inhibited constitutive autophagy, but not starvation-induced autophagy, as judged by the degradation of p62/SQSTM1, a substrate of autophagy [70]. The precise reason for this dysfunction is unknown, but it is possible to speculate that the active form of Rab33B recruits Atg5-12/16L complex to incorrect membranes because no obvious large LC3 dots were observed in cells expressing this mutant. Rab33B has another interaction partner, OATL1, a RabGAP, which can bind LC3 [71]. Rab33B seems to be a target substrate of OATL1 and to be involved in the fusion between autophagosomes and lysosomes. Overexpression of the wildtype or active form of Rab33B inhibits the fusion between autophagosomes and lysosomes, because OATL1 inactivates Rab33B as a GAP protein, and this inhibition leads to the blockade of autophagic flux. In fact, the expression of the active form of Rab33B increased the amount of LC3-II as previously reported [70], but did not show the colocalization of lysosomal membrane protein LAMP-1 with LC3-positive dots [71], suggesting that autophagosomes in the active form of Rab33B-expressing cells cannot efficiently fuse with lysosomes. Very recently, it was revealed that Atg16L interacts with Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L in neuroendocrine PC12 cells [72]. Knockdown of endogenous Atg16L in PC12 cells caused marked inhibition of hormone secretion inde‐ pendently of autophagic activity [72], indicating that Atg16L controls autophagy in all cell types as well as secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in neuroendocrine cells.

### **8. Conclusion**

phagosomal fusion with lysosomes [55]. Therefore, these results imply that Rab32 regulates

Recently, it has been reported that, in *Drosophila*, Rab32 colocalized with LysoTracker labeling lysosomes and GFP-Atg8 (LC3 homologue), indicating that Rab32 is localized at lysosomes and/or autophagosomes during programmed autophagy for metamorphosis to differentiate the fat body, salivary gland, and midgut [57]. Previously, Ma et al. reported that the Claret encoded by *claret*, a member of the granule group eye color genes [58], coprecipitated not only with Rab-RP1, a Rab GTPase encoded by *Drosophila lightoid*, but also with its human homo‐ logues, Rab32 and Rab38 [59]. Furthermore, the autophagosome formation was impaired in Rab32/*lightoid* mutants and Rab32 GEF/*claret* mutants, suggesting that Rab32 activity is required for the autophagic process of the fat body [57]. Previously, it has been suggested that autophagy impairment reduces lipid accumulation and impairs adipocytes differentiation in mice [60, 61]. In fact, downregulation of autophagy in *Drosophila* led to a decrease in the size of lipid droplets in *atg*-related genes in knocked down *Drosophila* fat body cells [57]; therefore, Rab32 appears to regulate lipid storage by controlling autophagy. Another report showed that Rab32 is upregulated in the epidermis and midgut during metamorphosis in *Helicoverpa*

*armigera* [62], suggesting that Rab32 may participate in organeogenesis in insects.

In addition to GEF, a GAP for Rab32, RUTBC1, was identified [63]. RUTBC1 is a TBC domaincontaining protein that binds to Rab9A in a nucleotide binding-state-dependent manner both in vitro and in vivo but has no GAP activity for Rab9A [63]; however, RUTBC1 acts as a GAP for Rab32 and Rab33B, and its TBC domain stimulates GTP hydrolysis [63]. Therefore, RUTBC1 may function in the autophagy process, as both Rab32 and Rab33B are suggested to be

Recently, *RAB32* and *IL23R (interleukin receptor 23)* were identified as susceptibility genes for leprosy in a genome-wide association study [64], although *IL23R* was previously reported to be a gene involved in Crohn's disease [65]. Leprosy, also known as Hansen's disease, is a chronic granulomatous infectious disease caused by *Mycobacterium leprae*, which affects both peripheral nerves and mucosa of the upper respiratory tract. As Rab32 participates in regu‐ lating the recruitment of cathepsin D to phagosomes containing *M. tuberculosis* [56], referred to above, Rab32 may have function in the pathogenesis of leprosy, such as host defense against

Rab33 has two isoforms, Rab33A and Rab33B. Rab33B is expressed ubiquitously, although Rab33A is expressed exclusively in the brain and cells of the immune system [66]. Rab33B is localized at the Golgi apparatus [67], although Rab33A also targets dense-core vesicles in

the recruitment of cathepsin D to the phagosomes.

**6.2. Rab32 GTPase and interaction molecules**

54 Autophagy - A Double-Edged Sword - Cell Survival or Death?

regulatory factors of autophagy.

**6.3. Rab32 GTPase and disease**

**7. Rab33 GTPase and autophagy**

*M. leprae* infection.

Based on the results of a large body of research, regulation of autophagy by Rab proteins could be loosely classified into two types, (i) autophagosome formation or (ii) autophagosome maturation (i.e. the fusion of autophagosomes with lysosomes) [Figure 2]. We assumed that Rab7, Rab9, and Rab11 could be sorted into type (ii) profile, and Rab24, Rab32 and Rab33 into type (i). Autophagy indicates not only the sequestration of materials into autophagosomes but also the degradation by lysosomal enzymes. Therefore, Rab proteins associated with the fusion step of autophagosomes with lysosomes have a fatal role in autophagic flux, and Rabs also trigger autophagosome formation. Type (ii)-categorized Rab proteins are mostly localized at late endosomes and play a role in membrane traffic to lysosomes, recycling endosomes or TGN. It is conceivable that they are involved in autophagosome maturation, because late endosomes, TGN, recycling endosomes, or Golgi-derived vesicles fuse with lysosomes routinely [73]. So far, no clear result has shown that Rab24 and Rab33 directly associate with the formation of autophagosomes except for Rab32, which is associated with ER and/or mitochondria and is regarded as a key factor for the supply to autophagosome formation [52]. However, it could be plausible that Rab33B is involved in autophagosome formation, since no obvious large LC3 dots were observed in the active form of Rab33B-expressing cells, but LC3 lipidation was induced [70]. Although the precise mechanism of autophagy by Rab24 is unknown, Rab24 might participate in the early stage of autophagy since overexpression of Rab24 stimulates the sequestration of *Coxiella* into vacuoles early after infection [47]. Thus, these Rab proteins, Rab24, Rab33, and Rab32, could be involved in autophagosome formation.

**Acknowledgements**

**Author details**

Yuko Hirota1

**References**

Sports, Sciences and Technology of Japan.

, Keiko Fujimoto2

Kyushu University, Maidashi, Fukuoka, Japan

ence 2000; 290(5497), 1717-1721.

lar Cell Biology 2009; 10(8), 513-525.

10(7), 458-467.

\*Address all correspondence to<EMAIL_ADDRESS>
cells. Cell Structure and Function 2002; 27(6), 421-429.

Annual Review of Biochemistry 2011; 80, 125-156.

glance. Journal of Cell Science 2012; 125. 2343-2348.

Sciences, Kyushu University, Maidashi, Fukuoka, Japan

This work was supported in part by a grant-in-aid from the Ministry of Education, Culture,

Rab GTPases in Autophagy http://dx.doi.org/10.5772/54578 57

and Yoshitaka Tanaka2\*

1 Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical

2 Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences,

[1] Klionsky DJ Emr SD. Autophagy as a regulated pathway of cellular degradation. Sci‐

[2] Mizushima N, Ohsumi Y, Yoshimori T. Autophagosome formation in mammalian

[3] Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nature Reviews Molecu‐

[4] Weidberg H, Shvets E, Elazar Z. Biogenesis and cargo selectivity of autophagosomes.

[5] Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autoph‐ agy mechanisms: lessons from yeast. Nature Reviews Molecular Cell Biology 2009;

[6] Shpilka T, Mizushima N, Elazar Z. Ubiquitin-like proteins and autophagy at a

[7] Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T. Dissection of autophagosome formation using Apg5-defi‐ cient mouse embryonic stem cells. Journal of Cell Biology 2001; 152(4), 657-668.

[8] Tanida I, Sou Y, Ezaki J, Minematsu-Ikeguchi N, Ueno T, Kominami E. HsAtg4B/ HsApg4B/Autophagin-1 cleaves the carboxyl termini of three human Atg8 homo‐

Although significant progress has been made in understanding the mechanisms of the formation and maturation of autophagic vacuoles through the function of Rab proteins, the precise molecular mechanism of how they regulate or interact with the core autophagic complex including Atg proteins is still unclear. Moreover, temporal ordering, such as a signal pathway to initiate the restricted molecules for autophagy, and spatial regulation including the source of isolation membranes are important in the overall understanding of autophagy involving Rab GTPases.

**Figure 2.** Localization and function of Rab GTPases for autophagy

## **Acknowledgements**

be plausible that Rab33B is involved in autophagosome formation, since no obvious large LC3 dots were observed in the active form of Rab33B-expressing cells, but LC3 lipidation was induced [70]. Although the precise mechanism of autophagy by Rab24 is unknown, Rab24 might participate in the early stage of autophagy since overexpression of Rab24 stimulates the sequestration of *Coxiella* into vacuoles early after infection [47]. Thus, these Rab proteins,

Although significant progress has been made in understanding the mechanisms of the formation and maturation of autophagic vacuoles through the function of Rab proteins, the precise molecular mechanism of how they regulate or interact with the core autophagic complex including Atg proteins is still unclear. Moreover, temporal ordering, such as a signal pathway to initiate the restricted molecules for autophagy, and spatial regulation including the source of isolation membranes are important in the overall understanding of autophagy

Rab24, Rab33, and Rab32, could be involved in autophagosome formation.

involving Rab GTPases.

56 Autophagy - A Double-Edged Sword - Cell Survival or Death?

**Figure 2.** Localization and function of Rab GTPases for autophagy

This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan.

## **Author details**

Yuko Hirota1 , Keiko Fujimoto2 and Yoshitaka Tanaka2\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi, Fukuoka, Japan

2 Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Fukuoka, Japan

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**Chapter 4**

**Flow Cytometric Measurement of**

N. Panchal, S. Chikte, B.R. Wilbourn, U.C. Meier and

The term autophagy, (Type II Apoptosis) is derived from the Greek roots "auto" (self) and "phagy" (eat) and was first coined by De Duve in 1967 to epitomise this type of cell death. The mechanism of organelle autophagy in cells undergoing macro-autophagy (from here on termed autophagy) is poorly understood. Cytoplasm, misfolded protein aggregates, dysfunc‐ tional mitochondria and stressed endoplasmic reticulum (ER) are engulfed by the formation of a double membrane forming an autophagosome [1,2]. The formation of the autophagosome double membrane structure within the cytoplasm is thought to be formed from pre-existing membranes within the cell, although it is unknown whether the Golgi apparatus, endoplasmic reticulum (ER) or mitochondria are used preferentially to form the autophagosome structure [1,2]. During the formation of the autophagosome structure, organelles such as mitochondria, parts of the ER and Golgi apparatus are engulfed by the autophagosome with the final closure of the double membrane structure occurring next. This then fuses with nearby lysosomes, giving rise to an autolysosome, where the intracellular components are degraded by hydrolytic enzymes [1,3-5]. This process generates ATP, which may delay cell death if the cell is under nutrient depleted conditions leading to the survival of the cell. Thus it is unclear whether the process protects or causes diseases such as cancer and neurodegenerative disorders [6,7].

The process of autophagy is a cell survival mechanism that occurs when the cell is under stress, via external environmental pressures, including the lack of nutrients, or via the internal microenvironment of the cell, including the replacement of old and defective organelles such as mitochondria [8,9]. Autophagy is also induced by the formation and collection of misfolded proteins in the endoplasmic reticulum (ER) which causes ER-stress within the cell [10]. Prolonged adverse conditions results in the death of the cell by the autophagic process. The

and reproduction in any medium, provided the original work is properly cited.

© 2013 Panchal et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Panchal et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

**Cell Organelle Autophagy**

G. Warnes

**1. Introduction**

http://dx.doi.org/10.5772/54711

## **Chapter 4**

## **Flow Cytometric Measurement of Cell Organelle Autophagy**

N. Panchal, S. Chikte, B.R. Wilbourn, U.C. Meier and G. Warnes

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54711

## **1. Introduction**

The term autophagy, (Type II Apoptosis) is derived from the Greek roots "auto" (self) and "phagy" (eat) and was first coined by De Duve in 1967 to epitomise this type of cell death. The mechanism of organelle autophagy in cells undergoing macro-autophagy (from here on termed autophagy) is poorly understood. Cytoplasm, misfolded protein aggregates, dysfunc‐ tional mitochondria and stressed endoplasmic reticulum (ER) are engulfed by the formation of a double membrane forming an autophagosome [1,2]. The formation of the autophagosome double membrane structure within the cytoplasm is thought to be formed from pre-existing membranes within the cell, although it is unknown whether the Golgi apparatus, endoplasmic reticulum (ER) or mitochondria are used preferentially to form the autophagosome structure [1,2]. During the formation of the autophagosome structure, organelles such as mitochondria, parts of the ER and Golgi apparatus are engulfed by the autophagosome with the final closure of the double membrane structure occurring next. This then fuses with nearby lysosomes, giving rise to an autolysosome, where the intracellular components are degraded by hydrolytic enzymes [1,3-5]. This process generates ATP, which may delay cell death if the cell is under nutrient depleted conditions leading to the survival of the cell. Thus it is unclear whether the process protects or causes diseases such as cancer and neurodegenerative disorders [6,7].

The process of autophagy is a cell survival mechanism that occurs when the cell is under stress, via external environmental pressures, including the lack of nutrients, or via the internal microenvironment of the cell, including the replacement of old and defective organelles such as mitochondria [8,9]. Autophagy is also induced by the formation and collection of misfolded proteins in the endoplasmic reticulum (ER) which causes ER-stress within the cell [10]. Prolonged adverse conditions results in the death of the cell by the autophagic process. The

© 2013 Panchal et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Panchal et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ER is responsible for the folding and then delivery of proteins via the secretory pathway to a functional site. Misfolded proteins accumulate in the lumen of the ER due to high protein folding demand on the ER [10]. Only properly folded proteins are secreted with maintenance of the plasma membrane structure and ER folding capacity being under ER homeostatic control [10,11]. Once a threshold of misfolded protein accumulation has been reached, a signal activates the ER to nucleus signalling pathway or the Unfolded Protein Response (UPR) causing ER chaperone proteins to be synthesised which refold the misfolded proteins and translocate these proteins to the cytoplasm for degradation by the proteasome [10,11]. This process results in an increase of the ER capacity to fold proteins and maintain ER homeostasis. This increase in ER capacity is physically achieved by an increase in size of the ER at early stages of autophagy [12]. If the protein folding demand continues to increase, this will ultimately result in the phagy of the ER itself which can be caused by ER stress (induced *in vitro* by tunicamycin and dithiothreitol, (DTT) and also multiple mechanisms that induce autophagy *e.g.* drugs such as rapamycin and nutrient starvation [10].

the cell cycle analysis doublet discrimination technique to accurately measure mitochondrial & ER mass during such treatments [16]. These techniques will serve as an adjunct to the measurement of autophagy microtubule associated protein, LC3I and LC3II (or LC3B as referred to from here on) in the determination of the presence of autophagy within a population

Flow Cytometric Measurement of Cell Organelle Autophagy

http://dx.doi.org/10.5772/54711

67

Methods for monitoring autophagy started with the initial discovery of the process by the use of electron microscopy showing the presence of double and single membrane structures termed the autophagosome and autolysosome or autophagolysosome respectively [4,5]. Other techniques have also obviously centred upon the formation of the autophagic machinery by measurement of LC3B, such as by Western Blotting which can be used to quantitate the degree of autophagy in cells by measuring LC3B which is normally located in the cytoplasm in the form of LC3I but when incorporated into the autophagosome is cleaved and lipidated by phosphotidylethanolamine, to form LC3II [17-19]. Anti-LC3B antibody labelling of LC3B has also been employed to measure autophagosomes and autolysosomes by image and flow cytometric analysis [20-22]. The increase in number and intensity of fluorescently labelled LC3B autophagosomes-autolysosomes can be quantitated by time-consuming image analysis, whereas increase in MFI of LC3B antigen levels measured flow cytometrically makes the process significantly less burdensome [21,23]. Here we describe a protocol to determine the presence of autophagy by the determination of LC3B levels in normal growing cells and those

Use of techniques to estimate the degree of loss of dysfunctional cell organelles has been more limited than those techniques investigating the development of the autophagic process. Previous studies have used ER Tracker and MitoTracker mass probes from Invitrogen to estimate ER and mitochondrial size based on median fluorescence intensity [16]. In experi‐ mental conditions different inducers of autophagy such as rapamycin, chloroquine, serum and total nutrient starvation may result in the different types of cell organelles being preferentially targeted by the autophagic process. Here we describe quantitative flow cytometric methods, which can be employed in the study of cell organelle-phagy. We show that a range of inducers cause the phagy of specific organelles and that this process is also cell type dependent. This article will highlight ways of monitoring the contribution of distinctive cell organelles in vitro

Jurkat T and K562 cell lines were grown in RMPI-1640 with L-glutamine (Cat No 21875-034, Invitrogen, Paisley, UK) supplemented with 10% foetal bovine serum (FBS, Cat No 10500-064, Invitrogen, Paisley, UK) and penicillin and streptomycin (Cat No 15140-122, Invitrogen,

C.

to the autophagic process and highlight its diversity in different cell types.

of cells.

undergoing autophagy.

**2. Materials and methods**

Paisley, UK) in the presence of 5% CO2 at 37o

**2.1. Cell lines**

Mitochondria are pivotal organelles in energy conversion. They act as the cell's power producers and are the site of cellular respiration, which ultimately generates fuel for the cell's activities. However, they are crucial for cell division, growth as well as cell death. As a major source of reactive oxygen species they consume cytosolic ATP when dysfunctional. It is, therefore pivotal for a cell to maintain of a cohort of healthy mitochondria. A sophisticated process called mitophagy, a selective process of autophagy, is responsible for the degradation of damaged organelles. In response to the triggers of mitophagy, mitochondria fragment, which sends out signals, which result in the engulfment by the autophagosomes [9,13]. Malfunctioning mitochondria are also generated through the process of aging or by having a high level of mutations in the mitochondrial DNA (mtDNA) induced by high levels of ROS. Mitochondrial DNA has 10-20 times more mutations than nuclear DNA [9]. Mitochondria reproduce by the process of fission, this produces two daughter mitochondria one of which hosts the damaged parts with reduced inner membrane potential of the original mitochondria but also a fully functional daughter [8]. The fission process can also stop excessive enlargement of mitochondria [8]. Conversely, mitochondrial fusion of damaged mitochondria dilutes the individual mitochondrion level of damaged macromolecules, and can prevent the early removal of such mitochondria [14]. Mitophagy is a physiological process which occurs during erythrocyte differentiation and during nutrient starvation [14]. The level of relative mitochon‐ drial fusion and fission within cells maintains mitochondrial homoeostasis. Thus mitophagy provides a mechanism by which aged or ROS damaged mitochondria are ultimately removed resulting in the survival of the cell in question.

Different agents/stimuli induce autophagy via different signalling routes and thus may preferentially cause mitophagy or if the ER is stressed by such stimuli, ER phagy or ER enlargement may be detectable at specific time points during each treatment. To this end we have employed organelle mass dyes to measure the relative size of mitochondria and ER during nutrient starvation, rapamycin, and chloroquine treatments. We have previously developed and modified the technique employed by Ramdzan *et al* [15] to measure linear scaled fluorescent signals of MitoTracker Green and ER Tracker Green via a modification of the cell cycle analysis doublet discrimination technique to accurately measure mitochondrial & ER mass during such treatments [16]. These techniques will serve as an adjunct to the measurement of autophagy microtubule associated protein, LC3I and LC3II (or LC3B as referred to from here on) in the determination of the presence of autophagy within a population of cells.

Methods for monitoring autophagy started with the initial discovery of the process by the use of electron microscopy showing the presence of double and single membrane structures termed the autophagosome and autolysosome or autophagolysosome respectively [4,5]. Other techniques have also obviously centred upon the formation of the autophagic machinery by measurement of LC3B, such as by Western Blotting which can be used to quantitate the degree of autophagy in cells by measuring LC3B which is normally located in the cytoplasm in the form of LC3I but when incorporated into the autophagosome is cleaved and lipidated by phosphotidylethanolamine, to form LC3II [17-19]. Anti-LC3B antibody labelling of LC3B has also been employed to measure autophagosomes and autolysosomes by image and flow cytometric analysis [20-22]. The increase in number and intensity of fluorescently labelled LC3B autophagosomes-autolysosomes can be quantitated by time-consuming image analysis, whereas increase in MFI of LC3B antigen levels measured flow cytometrically makes the process significantly less burdensome [21,23]. Here we describe a protocol to determine the presence of autophagy by the determination of LC3B levels in normal growing cells and those undergoing autophagy.

Use of techniques to estimate the degree of loss of dysfunctional cell organelles has been more limited than those techniques investigating the development of the autophagic process. Previous studies have used ER Tracker and MitoTracker mass probes from Invitrogen to estimate ER and mitochondrial size based on median fluorescence intensity [16]. In experi‐ mental conditions different inducers of autophagy such as rapamycin, chloroquine, serum and total nutrient starvation may result in the different types of cell organelles being preferentially targeted by the autophagic process. Here we describe quantitative flow cytometric methods, which can be employed in the study of cell organelle-phagy. We show that a range of inducers cause the phagy of specific organelles and that this process is also cell type dependent. This article will highlight ways of monitoring the contribution of distinctive cell organelles in vitro to the autophagic process and highlight its diversity in different cell types.

## **2. Materials and methods**

#### **2.1. Cell lines**

ER is responsible for the folding and then delivery of proteins via the secretory pathway to a functional site. Misfolded proteins accumulate in the lumen of the ER due to high protein folding demand on the ER [10]. Only properly folded proteins are secreted with maintenance of the plasma membrane structure and ER folding capacity being under ER homeostatic control [10,11]. Once a threshold of misfolded protein accumulation has been reached, a signal activates the ER to nucleus signalling pathway or the Unfolded Protein Response (UPR) causing ER chaperone proteins to be synthesised which refold the misfolded proteins and translocate these proteins to the cytoplasm for degradation by the proteasome [10,11]. This process results in an increase of the ER capacity to fold proteins and maintain ER homeostasis. This increase in ER capacity is physically achieved by an increase in size of the ER at early stages of autophagy [12]. If the protein folding demand continues to increase, this will ultimately result in the phagy of the ER itself which can be caused by ER stress (induced *in vitro* by tunicamycin and dithiothreitol, (DTT) and also multiple mechanisms that induce

Mitochondria are pivotal organelles in energy conversion. They act as the cell's power producers and are the site of cellular respiration, which ultimately generates fuel for the cell's activities. However, they are crucial for cell division, growth as well as cell death. As a major source of reactive oxygen species they consume cytosolic ATP when dysfunctional. It is, therefore pivotal for a cell to maintain of a cohort of healthy mitochondria. A sophisticated process called mitophagy, a selective process of autophagy, is responsible for the degradation of damaged organelles. In response to the triggers of mitophagy, mitochondria fragment, which sends out signals, which result in the engulfment by the autophagosomes [9,13]. Malfunctioning mitochondria are also generated through the process of aging or by having a high level of mutations in the mitochondrial DNA (mtDNA) induced by high levels of ROS. Mitochondrial DNA has 10-20 times more mutations than nuclear DNA [9]. Mitochondria reproduce by the process of fission, this produces two daughter mitochondria one of which hosts the damaged parts with reduced inner membrane potential of the original mitochondria but also a fully functional daughter [8]. The fission process can also stop excessive enlargement of mitochondria [8]. Conversely, mitochondrial fusion of damaged mitochondria dilutes the individual mitochondrion level of damaged macromolecules, and can prevent the early removal of such mitochondria [14]. Mitophagy is a physiological process which occurs during erythrocyte differentiation and during nutrient starvation [14]. The level of relative mitochon‐ drial fusion and fission within cells maintains mitochondrial homoeostasis. Thus mitophagy provides a mechanism by which aged or ROS damaged mitochondria are ultimately removed

Different agents/stimuli induce autophagy via different signalling routes and thus may preferentially cause mitophagy or if the ER is stressed by such stimuli, ER phagy or ER enlargement may be detectable at specific time points during each treatment. To this end we have employed organelle mass dyes to measure the relative size of mitochondria and ER during nutrient starvation, rapamycin, and chloroquine treatments. We have previously developed and modified the technique employed by Ramdzan *et al* [15] to measure linear scaled fluorescent signals of MitoTracker Green and ER Tracker Green via a modification of

autophagy *e.g.* drugs such as rapamycin and nutrient starvation [10].

66 Autophagy - A Double-Edged Sword - Cell Survival or Death?

resulting in the survival of the cell in question.

Jurkat T and K562 cell lines were grown in RMPI-1640 with L-glutamine (Cat No 21875-034, Invitrogen, Paisley, UK) supplemented with 10% foetal bovine serum (FBS, Cat No 10500-064, Invitrogen, Paisley, UK) and penicillin and streptomycin (Cat No 15140-122, Invitrogen, Paisley, UK) in the presence of 5% CO2 at 37o C.

#### **2.2. Induction of autophagy**

Jurkat and K562 cells were grown in <1% FBS-RPMI, <1% FBS-Phosphate Buffered Saline (PBS) (PBS, Cat No 14190-094 Invitrogen, Paisley, UK) or treated with chloroquine, (CQ) at 50µM, (Cat No C6628-25G, Sigma, Poole, UK), rapamycin (80nM, Cat No PHZ1233, Invitrogen, Paisley, UK). Time points analysed were 48h, (n=3) as described in the section below.

compensation was required. LC3B-Alexa Fluor-647 was detected on the 660/20nm channel on

Flow Cytometric Measurement of Cell Organelle Autophagy

http://dx.doi.org/10.5772/54711

69

Cells (10,000) were analysed on a Becton Dickinson FACSCanto II fitted with a 488nm Ti-Sapphire Argon laser, red HeNe 633nm laser and violet diode 405nm with FACSDiva Software ver 6.1.3. All data were analysed on FlowJo (ver 8.8.7, Treestar Inc, CA) in the form of list-mode data files version FCS 3.00 using the default bi-exponential transformation. Optical filters and

Student *t* tests were performed in Microsoft Office Excel with *P* = >0.05 not significant (NS), *P*

Cells treated to induce autophagy were first shown to be autophagic by immunofluorescent labelling of LC3B which is located in the autophagosome of cells undergoing autophagy. These were compared to resting control cells and positivity determine by use of an rabbit immuno‐ globulin isotype control labelled with the same secondary Alexa Fluor-647 conjugate permit‐ ting the demonstration that LC3B levels have been up-regulated in cell samples undergoing autophagy treatments. Figure 1 shows the LC3B signals from Jurkat and K562 cells after 48h of treatment compared to resting control levels. Resting cells do show a detectable LC3B compared to isotype controls (Figure 1). Jurkat cells showed a high level of LC3B compared to resting cells when treated with 80nM rapamycin and grown in low serum conditions (<1% FBS/RPMI, Figure 1A). In contrast CQ showed a lower level (half) of LC3B up-regulation compared to cells grown in nutrient free (<1% FBS) conditions (Figure 1B). K562 cell upregulation of LC3B was shown to be at a similar level of that of Jurkat cells, when treated with rapamycin and grown in low serum conditions (<1% FBS/RPMI, Figure 1C). CQ and nutrient free (<1% FBS) treatment of K562 cells showed a similar LC3B response to that of Jurkat cells

Rapamycin was shown to significantly up-regulate LC3B by 4-5 fold after 24 and 48h (P=<0.001, P=<0.005) in both cell lines (Figure 2A, B). In contrast low serum conditions caused a 5-6 fold increase in Jurkat LC3B only at 48h (P=<0.001, Figure 2A). Whilst, K562 responded to low serum treatment by a lower but significant 2-3 fold increase in LC3B levels at 24 and 48h (P=<0.005, P=<0.05, Figure 2B). Likewise nutrient depletion of K562 cells caused a 2 fold increase in LC3B at 24 and 48h (P=<0.01, P=NS, Figure 2B). Whilst nutrient depletion of Jurkat cells showed a significant 5-6 fold increase at 24h, reduced to a significant 2 fold increase above controls at 48h (P=<0.005, Figure 2A). CQ (50µM) in contrast to rapamycin induced a 50-100% increase in LC3B levels in Jurkat cells at 24 and 48h respectively (P=<0.05, P=NS, Figure 2A). Whilst K562 cells responded to CQ treatment (50µM) by doubling LC3B levels at 24h and falling back to

the Red HeNe trigon (BD FACSCanto II).

**2.6. Statistics**

**3. Results**

(Figure 1D).

mirrors in the BD FACSCanto II were fitted in 2008.

= <0.05\*, *P* = <0.01\*\*, *P* = <0.005\*\*\*, *P* = <0.001\*\*\*\*.

control levels at 48h respectively (P=NS, Figure 2B).

#### **2.3. Indirect immunofluorescence LC3B labeling**

Jurkat and K562 cells with or without treatment were pelleted and resuspended in 100 µl of Solution A fixative for 15 min at room temperature (RT) (Cat No GAS-002A-1, Caltag UK). Cells were then washed in PBS buffer Cell pellets were then permeabilised with 0.25% Triton X-100 (Cat No X100-500ML, Sigma Chemicals, Poole, UK) for 15 min at RT. Cells were washed in PBS buffer. Anti-LC3B polyclonal antibody (0.25 µg) (Cat No L10382, Invitrogen, Paisley, UK) or rabbit immunoglobulin (0.25 µg) (Cat No I5006, Sigma Chemicals, Poole, UK) was used as an isotype control and incubated for 0.5h at RT. Cells were then washed in PBS buffer. Cells were then labelled with 0.125 µg of secondary fluorescent conjugate Alexa Flour 647 goat antirabbit IgG (Cat No A21244, Invitrogen, Paisley, UK) for 30 min at RT. Cells were then washed in PBS buffer and resuspended in 400 µl of PBS. Analysis of LC3B-Alexa Fluor647 signal was achieved by determining the MFI of the whole histogram signal for previously gated cells from a FSC versus SSC dot-plot and compared to the corresponding isotype control sample in an overlaid histogram. 10,000 events were collected by flow cytometry.

#### **2.4. Organelle labelling**

Jurkat and K562 cells with or without treatment were counted and adjusted to the same number per volume and loaded with 40nM MitoTracker Green (MTG) (Cat No M7514, Invitrogen, Paisley, UK) or 100nM ER Tracker Green (ERTG) (Cat No L7526, Invitrogen, Paisley, UK) by incubating cells with dyes for 15 or 30 minutes at 37o C respectively. Cells were then washed in PBS buffer and resuspended in 400 µl of PBS, in the presence of DNA viability dye, DAPI (200 ng/ml) (Cat No D9542, Sigma Chemicals, Poole, UK). Live cells were analysed for MTG or ERTG MFI levels by firstly gating on all cell material except small debris in the origin of a FSC versus SSC dot-plot. This data was then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve. The 530/30nm channel on a BD FACS Canto II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot. Median fluorescence Intensity (MFI) of MTG-Area or ERTG-Area signals from samples was then compared by histogram analysis of untreated and treated cells.

#### **2.5. Flow cytometry**

Single colour controls for MTG, or ERTG and DAPI were used to set compensations. MTG or ERTG were detected in the 530/30nm channel on the argon laser octagon (BD FACSCanto II); DAPI was detected in the 440/40nm channel on the violet diode trigon (BD FACSCanto II). No compensation was required. LC3B-Alexa Fluor-647 was detected on the 660/20nm channel on the Red HeNe trigon (BD FACSCanto II).

Cells (10,000) were analysed on a Becton Dickinson FACSCanto II fitted with a 488nm Ti-Sapphire Argon laser, red HeNe 633nm laser and violet diode 405nm with FACSDiva Software ver 6.1.3. All data were analysed on FlowJo (ver 8.8.7, Treestar Inc, CA) in the form of list-mode data files version FCS 3.00 using the default bi-exponential transformation. Optical filters and mirrors in the BD FACSCanto II were fitted in 2008.

#### **2.6. Statistics**

**2.2. Induction of autophagy**

68 Autophagy - A Double-Edged Sword - Cell Survival or Death?

**2.4. Organelle labelling**

treated cells.

**2.5. Flow cytometry**

**2.3. Indirect immunofluorescence LC3B labeling**

overlaid histogram. 10,000 events were collected by flow cytometry.

incubating cells with dyes for 15 or 30 minutes at 37o

Jurkat and K562 cells were grown in <1% FBS-RPMI, <1% FBS-Phosphate Buffered Saline (PBS) (PBS, Cat No 14190-094 Invitrogen, Paisley, UK) or treated with chloroquine, (CQ) at 50µM, (Cat No C6628-25G, Sigma, Poole, UK), rapamycin (80nM, Cat No PHZ1233, Invitrogen,

Jurkat and K562 cells with or without treatment were pelleted and resuspended in 100 µl of Solution A fixative for 15 min at room temperature (RT) (Cat No GAS-002A-1, Caltag UK). Cells were then washed in PBS buffer Cell pellets were then permeabilised with 0.25% Triton X-100 (Cat No X100-500ML, Sigma Chemicals, Poole, UK) for 15 min at RT. Cells were washed in PBS buffer. Anti-LC3B polyclonal antibody (0.25 µg) (Cat No L10382, Invitrogen, Paisley, UK) or rabbit immunoglobulin (0.25 µg) (Cat No I5006, Sigma Chemicals, Poole, UK) was used as an isotype control and incubated for 0.5h at RT. Cells were then washed in PBS buffer. Cells were then labelled with 0.125 µg of secondary fluorescent conjugate Alexa Flour 647 goat antirabbit IgG (Cat No A21244, Invitrogen, Paisley, UK) for 30 min at RT. Cells were then washed in PBS buffer and resuspended in 400 µl of PBS. Analysis of LC3B-Alexa Fluor647 signal was achieved by determining the MFI of the whole histogram signal for previously gated cells from a FSC versus SSC dot-plot and compared to the corresponding isotype control sample in an

Jurkat and K562 cells with or without treatment were counted and adjusted to the same number per volume and loaded with 40nM MitoTracker Green (MTG) (Cat No M7514, Invitrogen, Paisley, UK) or 100nM ER Tracker Green (ERTG) (Cat No L7526, Invitrogen, Paisley, UK) by

in PBS buffer and resuspended in 400 µl of PBS, in the presence of DNA viability dye, DAPI (200 ng/ml) (Cat No D9542, Sigma Chemicals, Poole, UK). Live cells were analysed for MTG or ERTG MFI levels by firstly gating on all cell material except small debris in the origin of a FSC versus SSC dot-plot. This data was then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve. The 530/30nm channel on a BD FACS Canto II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot. Median fluorescence Intensity (MFI) of MTG-Area or ERTG-Area signals from samples was then compared by histogram analysis of untreated and

Single colour controls for MTG, or ERTG and DAPI were used to set compensations. MTG or ERTG were detected in the 530/30nm channel on the argon laser octagon (BD FACSCanto II); DAPI was detected in the 440/40nm channel on the violet diode trigon (BD FACSCanto II). No

C respectively. Cells were then washed

Paisley, UK). Time points analysed were 48h, (n=3) as described in the section below.

Student *t* tests were performed in Microsoft Office Excel with *P* = >0.05 not significant (NS), *P* = <0.05\*, *P* = <0.01\*\*, *P* = <0.005\*\*\*, *P* = <0.001\*\*\*\*.

## **3. Results**

Cells treated to induce autophagy were first shown to be autophagic by immunofluorescent labelling of LC3B which is located in the autophagosome of cells undergoing autophagy. These were compared to resting control cells and positivity determine by use of an rabbit immuno‐ globulin isotype control labelled with the same secondary Alexa Fluor-647 conjugate permit‐ ting the demonstration that LC3B levels have been up-regulated in cell samples undergoing autophagy treatments. Figure 1 shows the LC3B signals from Jurkat and K562 cells after 48h of treatment compared to resting control levels. Resting cells do show a detectable LC3B compared to isotype controls (Figure 1). Jurkat cells showed a high level of LC3B compared to resting cells when treated with 80nM rapamycin and grown in low serum conditions (<1% FBS/RPMI, Figure 1A). In contrast CQ showed a lower level (half) of LC3B up-regulation compared to cells grown in nutrient free (<1% FBS) conditions (Figure 1B). K562 cell upregulation of LC3B was shown to be at a similar level of that of Jurkat cells, when treated with rapamycin and grown in low serum conditions (<1% FBS/RPMI, Figure 1C). CQ and nutrient free (<1% FBS) treatment of K562 cells showed a similar LC3B response to that of Jurkat cells (Figure 1D).

Rapamycin was shown to significantly up-regulate LC3B by 4-5 fold after 24 and 48h (P=<0.001, P=<0.005) in both cell lines (Figure 2A, B). In contrast low serum conditions caused a 5-6 fold increase in Jurkat LC3B only at 48h (P=<0.001, Figure 2A). Whilst, K562 responded to low serum treatment by a lower but significant 2-3 fold increase in LC3B levels at 24 and 48h (P=<0.005, P=<0.05, Figure 2B). Likewise nutrient depletion of K562 cells caused a 2 fold increase in LC3B at 24 and 48h (P=<0.01, P=NS, Figure 2B). Whilst nutrient depletion of Jurkat cells showed a significant 5-6 fold increase at 24h, reduced to a significant 2 fold increase above controls at 48h (P=<0.005, Figure 2A). CQ (50µM) in contrast to rapamycin induced a 50-100% increase in LC3B levels in Jurkat cells at 24 and 48h respectively (P=<0.05, P=NS, Figure 2A). Whilst K562 cells responded to CQ treatment (50µM) by doubling LC3B levels at 24h and falling back to control levels at 48h respectively (P=NS, Figure 2B).

**Figure 1.** Demonstration of autophagy in Jurkat and K562 cells by measurement of LC3B levels by flow cytometry, see Materials & Methods. Resting Jurkat cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment with 80nM rapamycin and low serum/RPMI LC3B levels A). Resting Jurkat cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment of low serum/nutrient free conditions and CQ (50μM) LC3B levels B). Resting K562 cell LC3B-Alexa Flu‐ or-647 levels were compared after 48h treatment with 80nM rapamycin and low serum/RPMI LC3B levels C). Resting K562 cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment of low serum/nutrient free conditions and CQ (50μM) LC3B levels D).

**Figure 2.** Demonstration of autophagy in Jurkat and K562 cells by measurement of MFI LC3B levels by flow cytometry, see Materials & Methods. Resting Jurkat cell MFI LC3B-Alexa Fluor-647 levels were compared after 24 and 48h treat‐ ment with 80nM rapamycin, low serum/RPMI, low serum/nutrient free conditions and CQ (50μM) LC3B levels A). Rest‐ ing K562 cell MFI LC3B-Alexa Fluor-647 levels were compared after 24 and 48h treatment with 80nM rapamycin, low serum/RPMI, low serum/nutrient free conditions and CQ (50μM) LC3B levels A). T-test statistical analysis NS-not signif‐

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Likewise ER-phagy was determined by linear flow cytometric analysis of the ERTG signal in Jurkat and K562 cells by the following procedure.

Mitophagy was determined by linear flow cytometric analysis of the MTG signal in Jurkat and K562cellsbythefollowingprocedure.AftergatingoncellsfromaFSCvSSCdot-plot(Figure3A), livecellsweregateduponfromaFSCvDAPIdot-plot(Figure3B),DAPInegativecellsbeingalive. Doublet discrimination was determined from an MTG Area v Width parameter plot (Figure 3C) and MFI of control and test samples compared on an overlaid histogram (Figure 3D). In this example the Jurkat cell control gave an MFI of 142,000 compared to a 50µM CQ test showing an MFI of 100,000 or a 30% reduction in the mitochondrial mass of live Jurkat cells.

**Figure 2.** Demonstration of autophagy in Jurkat and K562 cells by measurement of MFI LC3B levels by flow cytometry, see Materials & Methods. Resting Jurkat cell MFI LC3B-Alexa Fluor-647 levels were compared after 24 and 48h treat‐ ment with 80nM rapamycin, low serum/RPMI, low serum/nutrient free conditions and CQ (50μM) LC3B levels A). Rest‐ ing K562 cell MFI LC3B-Alexa Fluor-647 levels were compared after 24 and 48h treatment with 80nM rapamycin, low serum/RPMI, low serum/nutrient free conditions and CQ (50μM) LC3B levels A). T-test statistical analysis NS-not signif‐ icant, \* P=<0.05, \*\* P=<0.01, \*\*\* P=<0.005, \*\*\*\* P=<0.001.

**Figure 1.** Demonstration of autophagy in Jurkat and K562 cells by measurement of LC3B levels by flow cytometry, see Materials & Methods. Resting Jurkat cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment with 80nM rapamycin and low serum/RPMI LC3B levels A). Resting Jurkat cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment of low serum/nutrient free conditions and CQ (50μM) LC3B levels B). Resting K562 cell LC3B-Alexa Flu‐ or-647 levels were compared after 48h treatment with 80nM rapamycin and low serum/RPMI LC3B levels C). Resting K562 cell LC3B-Alexa Fluor-647 levels were compared after 48h treatment of low serum/nutrient free conditions and

Mitophagy was determined by linear flow cytometric analysis of the MTG signal in Jurkat and K562cellsbythefollowingprocedure.AftergatingoncellsfromaFSCvSSCdot-plot(Figure3A), livecellsweregateduponfromaFSCvDAPIdot-plot(Figure3B),DAPInegativecellsbeingalive. Doublet discrimination was determined from an MTG Area v Width parameter plot (Figure 3C) and MFI of control and test samples compared on an overlaid histogram (Figure 3D). In this example the Jurkat cell control gave an MFI of 142,000 compared to a 50µM CQ test showing an

MFI of 100,000 or a 30% reduction in the mitochondrial mass of live Jurkat cells.

CQ (50μM) LC3B levels D).

70 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Likewise ER-phagy was determined by linear flow cytometric analysis of the ERTG signal in Jurkat and K562 cells by the following procedure.  Doublet discrimination was determined from an ERTG Area v Width parameter plot (Figure 4C) and MFI of control and test samples compared on an overlaid histogram (Figure 4D). In this example the K562 cell control gave an MFI of 159,000 compared to a 40nM rapamycin test showing an MFI of 262,000 or a 65% increase in the ER mass of live K562 cells.

**Figure 4.** K562 cells were untreated (control) or treated with 40nM rapamycin (R) for 48h. These cell cultures were then counted and adjusted to be the same number per volume and loaded with 100nM ERTG for 30 minutes at 37oC. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. Cells were then analysed on a BD Canto II according to Materials & Methods. Cells were gated upon except the small debris in the origin of a FSC versus SSC dot-plot (A). These data were then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve (B). The 530/30nm channel on a BD FACS Can‐ to II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot (C). The MFI of ERTG-Area signals from samples were then compared by histogram anal‐

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Induction of autophagy by the rapamycin mTOR signalling pathway induced a detectable mitophagy (34% reduction in mitochondrial mass) in Jurkat cells (Figure 5A). Whilst K562 cells displayed an ER stress response in that the ER mass was increased by 50% with rapamycin treatment without any mitophagy (Figure 5B). Like rapamycin, chloroquine induction of autophagy in Jurkat cells displayed a similar level of mitophagy (29%), whilst there was no significant organelle phagy displayed by K562 cells (Figure 5A, B). In contrast to rapamycin treatment of Jurkat cells, low serum/RPMI treatment caused a 19% increase in mitochondrial mass as opposed to a mitophagy. Under the same conditions Jurkat cells significantly increased

ysis of untreated, MFI 159,000 and rapamycin treated cells, MFI 262,000 (D).

**Figure 3.** Jurkat cells were untreated (control) or treated with 50μM CQ for 48h. These cell cultures were then count‐ ed and adjusted to be the same number per volume and loaded with 40nM MTG for 15 minutes at 37oC. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. Cells were then analysed on a BD Canto II ac‐ cording to Materials & Methods. Cells were gated upon except the small debris in the origin of a FSC versus SSC dotplot (A). There data were then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve (B). The 530/30nm channel on a BD FACS Canto II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot (C). The MFI of MTG-Area signals from samples were then compared by histogram analysis of untreated, MFI 142,000 and CQ treated cells, MFI 100,000 (D).

negative cells being alive. Doublet discrimination was determined from an ERTG Area v Width parameter plot (Figure 4C) and MFI of control and test samples compared on an overlaid histogram (Figure 4D). In this example the K562 cell control gave an MFI of 159,000 compared to a 40nM rapamycin test showing an MFI of 262,000 or a 65% increase in the ER mass of live

**Figure 3.** Jurkat cells were untreated (control) or treated with 50μM CQ for 48h. These cell cultures were then count‐ ed and adjusted to be the same number per volume and loaded with 40nM MTG for 15 minutes at 37oC. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. Cells were then analysed on a BD Canto II ac‐ cording to Materials & Methods. Cells were gated upon except the small debris in the origin of a FSC versus SSC dotplot (A). There data were then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve (B). The 530/30nm channel on a BD FACS Canto II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot (C). The MFI of MTG-Area signals from samples were then compared by histogram analysis of untreated, MFI 142,000 and CQ treated cells, MFI 100,000 (D).

K562 cells.

72 Autophagy - A Double-Edged Sword - Cell Survival or Death?

**Figure 4.** K562 cells were untreated (control) or treated with 40nM rapamycin (R) for 48h. These cell cultures were then counted and adjusted to be the same number per volume and loaded with 100nM ERTG for 30 minutes at 37oC. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. Cells were then analysed on a BD Canto II according to Materials & Methods. Cells were gated upon except the small debris in the origin of a FSC versus SSC dot-plot (A). These data were then analysed on a DAPI versus FSC dot-plot with live cells being DAPI-ve (B). The 530/30nm channel on a BD FACS Can‐ to II was set to linear and the width parameter activated. Doublet discrimination was then achieved by gating on single cells on a 530/30nm width and area plot (C). The MFI of ERTG-Area signals from samples were then compared by histogram anal‐ ysis of untreated, MFI 159,000 and rapamycin treated cells, MFI 262,000 (D).

Induction of autophagy by the rapamycin mTOR signalling pathway induced a detectable mitophagy (34% reduction in mitochondrial mass) in Jurkat cells (Figure 5A). Whilst K562 cells displayed an ER stress response in that the ER mass was increased by 50% with rapamycin treatment without any mitophagy (Figure 5B). Like rapamycin, chloroquine induction of autophagy in Jurkat cells displayed a similar level of mitophagy (29%), whilst there was no significant organelle phagy displayed by K562 cells (Figure 5A, B). In contrast to rapamycin treatment of Jurkat cells, low serum/RPMI treatment caused a 19% increase in mitochondrial mass as opposed to a mitophagy. Under the same conditions Jurkat cells significantly increased (37%, *P*<0.01) ER mass indicating a high level of misfolded proteins within the Jurkat cells (Figure 5A). However K562 cells responded in a similar manner to rapamycin treatment when treated with low serum/RPMI by displaying an increase in ER mass (43%) indicating again an autophagic response to a high level of misfolded proteins (Figure 5B). Nutrient depravation in the presence of low serum (<1%) showed the opposite response to low serum/RPMI treatment of cells in that a significant phagy of ER was observed in Jurkat cells (70%, *P*<0.05, Figure 5A). Whilst, K562 displayed both a significant ER phagy (42%) and mitophagy (36%), when undergoing nutrient depravation (*P*<0.05, Figure 5B).

**4. Discussion**

time point.

(similar to rapamycin) detected in Jurkat cells.

different organelle phagy in different cell types.

Modulation of autophagy may be important in preventing or treating neurodegenerative conditions and cancers. It is, therefore, pivotal to understand the underlying mechanisms. Numerous new techniques have been developed to show that cells are undergoing the autophagic process; these include LC3B semi-quantification by image and flow cytometric analysis by the use of GFP tagged proteins or antibody labelling [17-22,24-30]. Here we demonstrate by flow cytometric measurement of anti-LC3B-Alexa Fluor-647 signals that rapamycin significantly up-regulated LC3B in Jurkat and K562 cells, whilst CQ at 50µM, induced in comparison a small degree of detectable up-regulated LC3B in both cell lines employed in this study. Nutrient depletion studies, including reduced serum <1% and lack of nutrients induced a low level of LC3B up-regulation in K562 cells compared to the high level

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Different drugs or treatments induce autophagy via different signalling routes in different cell types resulting in varying types and degrees of organelle phagy. Rapamycin although induced a similar LC3B up-regulation in the two cell lines employed in this study resulted in different organelle phagy responses. Jurkat cells showed a mitophagy whilst K562 cells did not. This was in contrast to the lack of an effect of rapamycin upon Jurkat ER mass, whilst K562 cells showed an increase in ER mass, indicating protein mis-folding within the ER, even though rapamycin caused little cell death over the 48h period studied [10,11]. This increase in ER size after 48h of rapamycin treatment may ultimately result in a measurable ER-phagy at a later

Chloroquine induced apoptosis, death, low level autophagy (as indicated by mild LC3B upregulation) and mitophagy as indicated by the reduction in mitochondrial mass in Jurkat cells, whilst no organelles were targeted in K562 cells [31]. Thus both rapamycin and chloroquine act upon Jurkat cells to produce a mitophagy with no significant affect upon ER mass; whilst

Nutrient starvation commonly employed by removing serum from media in the study of autophagy caused protein mis-folding in both cell lines as indicated by an increase in ER mass and also of mitochondria in Jurkat cells [22]. In contrast total nutrient starvation which again caused varying degrees of up-regulation of LC3B levels in both cell lines as well as resulting in a large reduction in cell numbers caused a decrease in ER mass in both cell lines and a mitophagy in K562 cells. This again indicated that drug induction of autophagy may result in

Thus the combination of measuring the induction of autophagy via LC3B flow cytometric measurements and the technique of organelle mass semi-quantification gives the investigator more information about the autophagic process occurring within the cell not only in terms of autophagic flux signals but also the degree and type of organelle phagy occurring during the autophagic process. This technique of organelle mass semi-quantification by flow cytometry on live cells permits researchers in the field to measure not only the degree of autophagy but also live cell functions such as mitochondrial membrane potential during the autophagic

K562 cell response to these autophagy inducers was to show an increase in ER mass.

**Figure 5.** Jurkat (A) and K562 (B) cells were untreated (control) or treated 80nM rapamycin, low serum (<1% FBS) RPMI, nutrient free PBS (<1% FBS) or with 50μM CQ for 48h. These cell cultures were then counted and adjusted to be the same number per volume and loaded with 40nM MTG or 100nM ERTG for 15 or 30 minutes at 37oC respectively. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. The MFI of MTG or ERTG-Area signals from samples were then compared by histogram analysis of untreated or treated cells. T-test statistical analysis NS-not significant, \* P=<0.05, \*\* P=<0.01, \*\*\* P=<0.005, \*\*\*\* P=<0.001.

## **4. Discussion**

(37%, *P*<0.01) ER mass indicating a high level of misfolded proteins within the Jurkat cells (Figure 5A). However K562 cells responded in a similar manner to rapamycin treatment when treated with low serum/RPMI by displaying an increase in ER mass (43%) indicating again an autophagic response to a high level of misfolded proteins (Figure 5B). Nutrient depravation in the presence of low serum (<1%) showed the opposite response to low serum/RPMI treatment of cells in that a significant phagy of ER was observed in Jurkat cells (70%, *P*<0.05, Figure 5A). Whilst, K562 displayed both a significant ER phagy (42%) and mitophagy (36%),

**Figure 5.** Jurkat (A) and K562 (B) cells were untreated (control) or treated 80nM rapamycin, low serum (<1% FBS) RPMI, nutrient free PBS (<1% FBS) or with 50μM CQ for 48h. These cell cultures were then counted and adjusted to be the same number per volume and loaded with 40nM MTG or 100nM ERTG for 15 or 30 minutes at 37oC respectively. Cells were washed and adjusted to 0.5ml volume containing DAPI at 200ng/ml. The MFI of MTG or ERTG-Area signals from samples were then compared by histogram analysis of untreated or treated cells. T-test statistical analysis NS-not

significant, \* P=<0.05, \*\* P=<0.01, \*\*\* P=<0.005, \*\*\*\* P=<0.001.

when undergoing nutrient depravation (*P*<0.05, Figure 5B).

74 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Modulation of autophagy may be important in preventing or treating neurodegenerative conditions and cancers. It is, therefore, pivotal to understand the underlying mechanisms. Numerous new techniques have been developed to show that cells are undergoing the autophagic process; these include LC3B semi-quantification by image and flow cytometric analysis by the use of GFP tagged proteins or antibody labelling [17-22,24-30]. Here we demonstrate by flow cytometric measurement of anti-LC3B-Alexa Fluor-647 signals that rapamycin significantly up-regulated LC3B in Jurkat and K562 cells, whilst CQ at 50µM, induced in comparison a small degree of detectable up-regulated LC3B in both cell lines employed in this study. Nutrient depletion studies, including reduced serum <1% and lack of nutrients induced a low level of LC3B up-regulation in K562 cells compared to the high level (similar to rapamycin) detected in Jurkat cells.

Different drugs or treatments induce autophagy via different signalling routes in different cell types resulting in varying types and degrees of organelle phagy. Rapamycin although induced a similar LC3B up-regulation in the two cell lines employed in this study resulted in different organelle phagy responses. Jurkat cells showed a mitophagy whilst K562 cells did not. This was in contrast to the lack of an effect of rapamycin upon Jurkat ER mass, whilst K562 cells showed an increase in ER mass, indicating protein mis-folding within the ER, even though rapamycin caused little cell death over the 48h period studied [10,11]. This increase in ER size after 48h of rapamycin treatment may ultimately result in a measurable ER-phagy at a later time point.

Chloroquine induced apoptosis, death, low level autophagy (as indicated by mild LC3B upregulation) and mitophagy as indicated by the reduction in mitochondrial mass in Jurkat cells, whilst no organelles were targeted in K562 cells [31]. Thus both rapamycin and chloroquine act upon Jurkat cells to produce a mitophagy with no significant affect upon ER mass; whilst K562 cell response to these autophagy inducers was to show an increase in ER mass.

Nutrient starvation commonly employed by removing serum from media in the study of autophagy caused protein mis-folding in both cell lines as indicated by an increase in ER mass and also of mitochondria in Jurkat cells [22]. In contrast total nutrient starvation which again caused varying degrees of up-regulation of LC3B levels in both cell lines as well as resulting in a large reduction in cell numbers caused a decrease in ER mass in both cell lines and a mitophagy in K562 cells. This again indicated that drug induction of autophagy may result in different organelle phagy in different cell types.

Thus the combination of measuring the induction of autophagy via LC3B flow cytometric measurements and the technique of organelle mass semi-quantification gives the investigator more information about the autophagic process occurring within the cell not only in terms of autophagic flux signals but also the degree and type of organelle phagy occurring during the autophagic process. This technique of organelle mass semi-quantification by flow cytometry on live cells permits researchers in the field to measure not only the degree of autophagy but also live cell functions such as mitochondrial membrane potential during the autophagic process giving an insight to the more precise mechanism of action of the wide variety of stimuli that can be employed in the study of the autophagic process. This area warrants further study as it holds new therapeutic and diagnostic potential.

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Death and Differentiation 2007;15:171-182.

## **Author details**

N. Panchal1 , S. Chikte1 , B.R. Wilbourn1 , U.C. Meier2 and G. Warnes1

1 Flow Cytometry Core Facility, The Blizard Institute, Barts and The London School of Med‐ icine and Dentistry, Queen Mary London University, UK

2 Centre for Neuroscience and Trauma, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary London University, London, UK

### **References**

[10] Yorimitsu T, Klionsky DJ. Eating the endoplasmic reticulum: quality control by au‐ tophagy. Trends in Cell Biology 2007;17:279-285.

process giving an insight to the more precise mechanism of action of the wide variety of stimuli that can be employed in the study of the autophagic process. This area warrants further study

, U.C. Meier2

1 Flow Cytometry Core Facility, The Blizard Institute, Barts and The London School of Med‐

2 Centre for Neuroscience and Trauma, The Blizard Institute, Barts and The London School

[1] Tooze S, Yoshimori T. The origin of the autophagosomal membrane. Nature Cell Bi‐

[2] Mehrpour M, Esclatine A, Beau I, Codogno P. Overview of macroautophagy regula‐

[3] Yang Z, Klionsky D. Eaten alive: a history of macroautophagy. Nature Cell Biology

[4] Ashford TP, Porter K. Cytoplasmic components in hepatic cell lysosomes. J Cell Biol

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[6] Shintani T, Klionsky D. Autophagy in health and disease: A double-edged sword.

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**Section 2**

**Consequences of Autophagy Deficits**

**Consequences of Autophagy Deficits**

[24] Shvets E, Fass E, Elazar Z. Utlizing flow cytometry to monitor autophagy in living

[25] Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation proc‐ ess by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy

[26] Wu YT, Tan HL, Huang Q, Kim YS, Pan N, Ong WY, Liu ZG, Ong CN, Shen HM. Autophagy plays a protective role during zVAD-induced necrotic cell death. Au‐

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[28] Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Metivier D, Meley D, Souquere S, Yoshimori T and others. Inhibition of macroautophagy trig‐

[29] Byun JY, Yoon CH, An S, Park IC, Kang CM, Kim MJ, SJ. L. The Rac1/MKK7/JNK pathway signals upregulation of Atg5 and subsequent autophagic cell death in re‐

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Autophagy 2009;5:964-972.

**Chapter 5**

**Autophagy, the "Master" Regulator of Cellular Quality**

Autophagy an evolutionarily conserved process, is activated in response to nutrient depriva‐ tion, as well as to endogenous and exogenous stresses. It is currently known that the basic mechanism of autophagy has been well conserved during evolution since diverse organisms, including yeast, flies, and mammals, all carry a similar set of autophagy-related genes (ATGs), although there are some significant differences between yeast and higher eukaryotes. As a result, autophagy is a highly regulated process known to contribute to cellular cleaning through the removal of intracellular components in lysosomes showing therefore an important role in cellular quality control. Autophagy ensures that proteins damaged or incorrectly synthesized are removed from the cells by degradation, preventing thus the devastating cellular consequences associated with accumulation of malfunctioning proteins inside cells. Moreover autophagy works as a recycling system where it mediates the breakdown of proteins that are no longer needed into essential components (aminoacids, free fatty acids, sugars), which can then be used in the synthesis of new proteins. This has extreme importance in conditions of nutritional stress or starvation. At optimal physiological conditions in the absence of stressors, basal level of autophagy assures maintenance of cell homeostasis through regular turnover of proteins, lipids, and organelles. Therefore autophagy acts a cautious controller of cellular homeostasis. In addition, it also has an important role in cellular defence as in compromised situations it contributes to the proteolytic breakdown of components of invading pathogens and other types of biological cell aggressors. Autophagy may also modulate synaptic plasticity, which involves structural remodelling of nerve terminals and the trafficking and degradation of receptors and other synaptic proteins [1]. Finally, autophagy is also a key player in cellular adaptation since is able of changing very rapidly the rate of a

> © 2013 Esteves et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Esteves et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Control: What Happens when Autophagy Fails?**

A. Raquel Esteves, Catarina R. Oliveira and

Additional information is available at the end of the chapter

Sandra Morais Cardoso

http://dx.doi.org/10.5772/55196

**1. Introduction**

**Chapter 5**

## **Autophagy, the "Master" Regulator of Cellular Quality Control: What Happens when Autophagy Fails?**

A. Raquel Esteves, Catarina R. Oliveira and Sandra Morais Cardoso

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55196

### **1. Introduction**

Autophagy an evolutionarily conserved process, is activated in response to nutrient depriva‐ tion, as well as to endogenous and exogenous stresses. It is currently known that the basic mechanism of autophagy has been well conserved during evolution since diverse organisms, including yeast, flies, and mammals, all carry a similar set of autophagy-related genes (ATGs), although there are some significant differences between yeast and higher eukaryotes. As a result, autophagy is a highly regulated process known to contribute to cellular cleaning through the removal of intracellular components in lysosomes showing therefore an important role in cellular quality control. Autophagy ensures that proteins damaged or incorrectly synthesized are removed from the cells by degradation, preventing thus the devastating cellular consequences associated with accumulation of malfunctioning proteins inside cells. Moreover autophagy works as a recycling system where it mediates the breakdown of proteins that are no longer needed into essential components (aminoacids, free fatty acids, sugars), which can then be used in the synthesis of new proteins. This has extreme importance in conditions of nutritional stress or starvation. At optimal physiological conditions in the absence of stressors, basal level of autophagy assures maintenance of cell homeostasis through regular turnover of proteins, lipids, and organelles. Therefore autophagy acts a cautious controller of cellular homeostasis. In addition, it also has an important role in cellular defence as in compromised situations it contributes to the proteolytic breakdown of components of invading pathogens and other types of biological cell aggressors. Autophagy may also modulate synaptic plasticity, which involves structural remodelling of nerve terminals and the trafficking and degradation of receptors and other synaptic proteins [1]. Finally, autophagy is also a key player in cellular adaptation since is able of changing very rapidly the rate of a

© 2013 Esteves et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Esteves et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

particular protein's degradation allowing fast modulation of the proteome in response to stress. The lysosomal system and specifically the autophagic pathway is the principal mech‐ anism for degradating longer-lived proteins and is the only system in cells capable of degrad‐ ing organelles and protein aggregates/inclusions. Similar to what happens with the proteasome system, protein aggregates and certain organelles have been shown to be tagged with ubiquitination for selective removal by autophagy [2]. An adaptor molecule with an ubiquitin-binding domain engages the ubiquitinated structure and couples it to the preautophagosomal isolation membrane for subsequent sequestration. The exact types of ubiquitin motifs recognized by the proteasome and autophagy may differ and the degree to which ubiquitination drives autophagic protein turn-over relative to that by proteasomes is still unclear.

degraded. Microautophagy participates in the continuous, slow "basal" turnover of cellular components in normal cellular conditions [10]. Furthermore microautophagy has been shown to be responsible for the selective removal of organelles when they are no longer needed [11]. The absence of mammalian homologs for the microautophagy yeast genes has made it difficult

Autophagy, the "Master" Regulator of Cellular Quality Control: What Happens when Autophagy Fails?

http://dx.doi.org/10.5772/55196

83

The third type of mammalian autophagy CMA distinguishes from the others because of its distinctive feature of selectivity. Unlike the other forms of autophagy, in which portions of the cytoplasm are typically engulfed, proteins degraded by CMA are identified and transported one-by-one by a cytosolic chaperone that delivers them to the surface of the lysosomes. First there is individual recognition of single proteins in the citosol, then the substrate proteins unfold and cross the lysosomal membrane. Only soluble proteins containing in their aminoacid sequence a pentapeptide motif related to KFERQ, are recognized by heat shock cognate protein of 70 kDa, hsc70, which mediates their delivery to the lysosomes for direct translocation across the lysosomal membrane [3, 12]. These pentapeptide motifs are recognized specifically by the cytosolic protein hsc70, the constitutively expressed member of the hsp70 family of cytosolic chaperones. hsc70 not only targets the CMA substract to the lysosomal membrane where it can interact with the CMA receptor but also facilitates substrate folding [13]. A second chaperone that also localizes on both sides of the lysosomal membrane is the heat shock protein of 90 kDa, hsp90. hsp90 is proposed to participate in substrate protein unfolding [14], and contributes to the stabilization of essential components of the translocation complex as they organize into a multimeric structure [15]. Additionally, the co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) has been shown to mediate lysosomal degradation of proteins such as alpha-synuclein, a known CMA substrate [16], however, it is still undergoing investigation whether CHIP-mediated degradation occurs via CMA or via other forms of autophagy. The lysosome-associated membrane protein type 2A (LAMP-2A) acts as a receptor for substrates of this pathway [17]. This protein is one of the three splice variants encoded by the lamp2 gene that share identical lumenal regions and different transmembrane and cytosolic tails [18]. Once the cytosolic proteins destined for CMA degradation bind to the cytosolic tail of the single-span membrane protein LAMP-2A, they promote its assembly into a high molecular weight complex of about 700 kDa at the lysosomal membrane required for translocation of the CMA substrates into the lysosomal lumen [15]. Multimerization is a required step for substrate translocation. However, the translocation complex is not stable at the lysosomal membrane and, once the substrate reaches the lysosomal lumen, LAMP-2A dissociates into monomers. hsc70 mediates the disassembly generating monomeric forms of LAMP-2A that are required for substrate binding in order to sustain cycles of binding and uptake. Other proteins have been shown to modulate the stability of the CMA translocation complex, such as, glial fibrillary acidic protein (GFAP) and elongation factor 1 a (EF1a). One of the crucial functions of CMA is protein quality control via the selective degradation of damaged or altered proteins. Moreover proteins associated with several neurodegeneratives disorders have KFERQ-like sequences such as, alpha-synuclein, parkin, UCHL1 and Pink1, amyloid precursor protein (APP) and tau and huntingtin. These proteins can be degraded by CMA only when they are in soluble forms; once the insoluble inclusions are formed, they can

in gaining a better understanding of the pathophysiology of this process.

There are three basic forms of autophagy, namely, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), which primarily differ in the way in which cytosolic components are delivered to lysosomes [3]. Moreover, the different types of autophagy share a common endpoint, the lysosome, but differ in the substrates targeted, their regulation, and the conditions in which each of them is preferentially activated.

In macroautophagy and microautophagy there is the sequestration of cytoplasmic components into vesicular compartments. In the case of macroautophagy, vesicles are form *de novo* from a limiting membrane of non-lysosomal origin that closes and forms a double membrane organelle called autophagosome [4]. Because the autophagosome lacks any enzymes, the trapped contents are not degraded until the autophagosome fuses with a lysosome, which provides all the hydrolases required for degradation of cargo. Autophagosomes are then transported along microtubules to the perinuclear region of the cell (where lysosomes are clustered) to enhance the probability of autophagosome-lysosome fusion to form autophago‐ lysosomes. Lysosomes are single membrane organelles dedicated to degradation of both intracellular and extracellular components. Lysosomal membranes possess vacuolar H+ ATPases which are proton pumps that acidificate the autophagolysosome contents. This acidification is essential for the activation of lysosomal enzymes (including proteases, lipases, glycosidases, and nucleases), which are responsible for proteolysis of the components in the autophagolysosome and confer this organelle its high degradative capacity [5]. Although autophagy was initially considered to be a nonselective process, cargo-specific autophagy (the selective degradation of certain aggregated proteins and organelles) is now recognized to exist [6]. Numerous different types of cargo-specific autophagic processes have been described, like for instance mitophagy where damaged mitochondria are sequestered and degraded [7]. Macroautophagy occurs constitutively in cells and is markedly induced under stress condi‐ tions, such as starvation and has two major purposes: as a source to generate essential macromolecules and energy in conditions of nutritional scarcity, or as a mechanism for the removal of altered intracellular components [8].

In the case of microautophagy the engulfment of cargo occurs through invagination of the lysosomal membrane itself, to form vesicles that invaginate towards the lysosomal lumen [9]. Cytosolic regions are sequestered directly by the lysosome through invaginations or tubula‐ tions that "pinch off" from the membrane into the lysosomal lumen where they are rapidly degraded. Microautophagy participates in the continuous, slow "basal" turnover of cellular components in normal cellular conditions [10]. Furthermore microautophagy has been shown to be responsible for the selective removal of organelles when they are no longer needed [11]. The absence of mammalian homologs for the microautophagy yeast genes has made it difficult in gaining a better understanding of the pathophysiology of this process.

particular protein's degradation allowing fast modulation of the proteome in response to stress. The lysosomal system and specifically the autophagic pathway is the principal mech‐ anism for degradating longer-lived proteins and is the only system in cells capable of degrad‐ ing organelles and protein aggregates/inclusions. Similar to what happens with the proteasome system, protein aggregates and certain organelles have been shown to be tagged with ubiquitination for selective removal by autophagy [2]. An adaptor molecule with an ubiquitin-binding domain engages the ubiquitinated structure and couples it to the preautophagosomal isolation membrane for subsequent sequestration. The exact types of ubiquitin motifs recognized by the proteasome and autophagy may differ and the degree to which ubiquitination drives autophagic protein turn-over relative to that by proteasomes is

There are three basic forms of autophagy, namely, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), which primarily differ in the way in which cytosolic components are delivered to lysosomes [3]. Moreover, the different types of autophagy share a common endpoint, the lysosome, but differ in the substrates targeted, their regulation, and

In macroautophagy and microautophagy there is the sequestration of cytoplasmic components into vesicular compartments. In the case of macroautophagy, vesicles are form *de novo* from a limiting membrane of non-lysosomal origin that closes and forms a double membrane organelle called autophagosome [4]. Because the autophagosome lacks any enzymes, the trapped contents are not degraded until the autophagosome fuses with a lysosome, which provides all the hydrolases required for degradation of cargo. Autophagosomes are then transported along microtubules to the perinuclear region of the cell (where lysosomes are clustered) to enhance the probability of autophagosome-lysosome fusion to form autophago‐ lysosomes. Lysosomes are single membrane organelles dedicated to degradation of both intracellular and extracellular components. Lysosomal membranes possess vacuolar H+ ATPases which are proton pumps that acidificate the autophagolysosome contents. This acidification is essential for the activation of lysosomal enzymes (including proteases, lipases, glycosidases, and nucleases), which are responsible for proteolysis of the components in the autophagolysosome and confer this organelle its high degradative capacity [5]. Although autophagy was initially considered to be a nonselective process, cargo-specific autophagy (the selective degradation of certain aggregated proteins and organelles) is now recognized to exist [6]. Numerous different types of cargo-specific autophagic processes have been described, like for instance mitophagy where damaged mitochondria are sequestered and degraded [7]. Macroautophagy occurs constitutively in cells and is markedly induced under stress condi‐ tions, such as starvation and has two major purposes: as a source to generate essential macromolecules and energy in conditions of nutritional scarcity, or as a mechanism for the

In the case of microautophagy the engulfment of cargo occurs through invagination of the lysosomal membrane itself, to form vesicles that invaginate towards the lysosomal lumen [9]. Cytosolic regions are sequestered directly by the lysosome through invaginations or tubula‐ tions that "pinch off" from the membrane into the lysosomal lumen where they are rapidly

the conditions in which each of them is preferentially activated.

82 Autophagy - A Double-Edged Sword - Cell Survival or Death?

removal of altered intracellular components [8].

still unclear.

The third type of mammalian autophagy CMA distinguishes from the others because of its distinctive feature of selectivity. Unlike the other forms of autophagy, in which portions of the cytoplasm are typically engulfed, proteins degraded by CMA are identified and transported one-by-one by a cytosolic chaperone that delivers them to the surface of the lysosomes. First there is individual recognition of single proteins in the citosol, then the substrate proteins unfold and cross the lysosomal membrane. Only soluble proteins containing in their aminoacid sequence a pentapeptide motif related to KFERQ, are recognized by heat shock cognate protein of 70 kDa, hsc70, which mediates their delivery to the lysosomes for direct translocation across the lysosomal membrane [3, 12]. These pentapeptide motifs are recognized specifically by the cytosolic protein hsc70, the constitutively expressed member of the hsp70 family of cytosolic chaperones. hsc70 not only targets the CMA substract to the lysosomal membrane where it can interact with the CMA receptor but also facilitates substrate folding [13]. A second chaperone that also localizes on both sides of the lysosomal membrane is the heat shock protein of 90 kDa, hsp90. hsp90 is proposed to participate in substrate protein unfolding [14], and contributes to the stabilization of essential components of the translocation complex as they organize into a multimeric structure [15]. Additionally, the co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) has been shown to mediate lysosomal degradation of proteins such as alpha-synuclein, a known CMA substrate [16], however, it is still undergoing investigation whether CHIP-mediated degradation occurs via CMA or via other forms of autophagy. The lysosome-associated membrane protein type 2A (LAMP-2A) acts as a receptor for substrates of this pathway [17]. This protein is one of the three splice variants encoded by the lamp2 gene that share identical lumenal regions and different transmembrane and cytosolic tails [18]. Once the cytosolic proteins destined for CMA degradation bind to the cytosolic tail of the single-span membrane protein LAMP-2A, they promote its assembly into a high molecular weight complex of about 700 kDa at the lysosomal membrane required for translocation of the CMA substrates into the lysosomal lumen [15]. Multimerization is a required step for substrate translocation. However, the translocation complex is not stable at the lysosomal membrane and, once the substrate reaches the lysosomal lumen, LAMP-2A dissociates into monomers. hsc70 mediates the disassembly generating monomeric forms of LAMP-2A that are required for substrate binding in order to sustain cycles of binding and uptake. Other proteins have been shown to modulate the stability of the CMA translocation complex, such as, glial fibrillary acidic protein (GFAP) and elongation factor 1 a (EF1a). One of the crucial functions of CMA is protein quality control via the selective degradation of damaged or altered proteins. Moreover proteins associated with several neurodegeneratives disorders have KFERQ-like sequences such as, alpha-synuclein, parkin, UCHL1 and Pink1, amyloid precursor protein (APP) and tau and huntingtin. These proteins can be degraded by CMA only when they are in soluble forms; once the insoluble inclusions are formed, they can only be degraded via other proteolytic pathways [19]. CMA is constitutively active in many cell types and similar to macroautophagy, CMA is maximally activated under stress conditions like nutritional stress or starvation and cellular stresses leading to protein damage [3]. Indeed, upon stressors that cause protein damage, such as oxidative stress, CMA is upregulated [20]. During starvation, macroautophagy is first activated, and if starvation persists, cells activate CMA, which selectively targets non-essential proteins for degradation to obtain the aminoa‐ cids required for the synthesis of essential proteins showing that macroautophagy and CMA act in a synchronized manner [21]. There are two lysosomal components that limit CMA activity: LAMP-2A and lys-hsc70. It is known that changes in the number of LAMP-2A molecules, as well as, in the membrane content of LAMP-2A, rapidly upregulate or downre‐ gulate CMA activity [17]. Levels of lys-hsc70 increase gradually with the increase in CMA activity, although the mechanisms modulating this increase are still poorly understood [14]. Furthermore, the activity of this autophagic pathway is also directly modulated by changes in other autophagic and proteolytic systems inside the cell. For examples, cells in culture respond to CMA blockage by upregulating macroautophagy [21]. Likewise, blockage of macroautoph‐ agy results in constitutive activation of CMA [22].

Autophagy can be regulated by mTOR-dependent signaling pathways and mTOR-independ‐ ent signaling pathways. The serine–threonine kinase mTOR is a master negative regulator of autophagy that acts by blocking the activity of the ULK1/Atg1 complex [26]. mTOR is inhibited when nutrients are scarce, when growth factor signaling is reduced, and under ATP depletion. When mTOR is inhibited and the repressive effect of mTOR on autophagosome formation disappears an increase in autophagosome biogenesis occurs [27]. In pathways that act independently of mTOR, when in nutrient-rich conditions, Beclin-1 is bound to the antiapop‐ totic protein Bcl-2. Under nutrient starvation, the stress-activated enzyme Jun N-terminal kinase 1 (JNK1) phosphorylates Bcl-2, which induces its dissociation from Beclin-1 [28]. Beclin can then interact with other members of the autophagic machinery and stimulate the induction of autophagy [29-30]. One of the mTOR-independent pathways can be mediated by the IP3 pathway and the Ca2+–calpain–G-stimulatory protein alpha pathway [31]. Generation of IP3 induces the release of Ca2+ from stores in the endoplasmic reticulum and high levels of cytosolic Ca2+ inhibit autophagy by activating calpains. After cleavage by calpains, the Ca2+–calpain–Gstimulatory protein alpha pathway becomes activated which, in turn, causes the production of more inhibitory cAMP [31]. On the other hand, decreased levels of IP3 signaling leads to reduced Ca2+ release from the endoplasmic reticulum and lower rates of mitochondrial Ca2+ uptake, causing a reduction in mitochondrial activity and ATP depletion, which results in 5'- AMP-activated protein kinase (AMPK) activation [32]. Activated AMPK directly phosphory‐ lates ULK1, indirectly inactivating mTOR complex which leads to the induction of autophagy.

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85

Two of the main aggravating factors contributing to failure of autophagic pathways are believed to be oxidative stress and aging. Aging can lead to reductions in autophagosome formation and autophagosome-lysosome fusion [33]. Moreover leads to lysosomal alterations such as increased lysosome volume, decreased lysosomal stability, altered activity of hydro‐ lases and intralysosomal accumulation of indigestible material such as lipofuscin [34]. These alterations are consistent with a decrease in autophagy, more specifically macroautophagy. Consequently, there is a reduction in the turnover of intracellular components as well as a reduction in the ability of cells to adapt to changes in the extracellular environment [35]. Ultimately this contributes to the intracellular accumulation of misfolded proteins in aged organisms [36]. Moreover, also a decline in CMA activity has been described in almost all cell types and tissues. This decrease is primarily due to a reduction in the levels of LAMP-2A at the lysosomal membrane [37]. In addition, the stability of LAMP-2A at the lysosomal mem‐ brane is markedly reduced with age, resulting in a decrease in the net content of this protein

On the other hand, oxidative stress overloads the macroautophagic system, and oxidized proteins and damaged organelles engulfed by autophagosomes can become a source of ROS inside either autophagosomes or lysosomes. Subsequently ROS can react and damage lysosomal hydrolases and/or other components required for the lysosome/autophagosome fusion, resulting in the accumulation of undegraded products inside these cellular compart‐

**2. Autophagic failure at cellular level**

in lysosomes [38].

The identification of ATGs in yeast was a major breakthrough in the study of autophagy [23]. Until now more than 35 ATG genes have been found in yeast and many of them have orthologs in mammals to control the dynamic processes and different stages of autophagy. Indeed, autophagic activities are mediated by a complex molecular machinery including more than 35 ATG-related proteins and 50 lysosomal hydrolases. This dynamic macroautophagic process includes initiation, nucleation, elongation, maturation and degradation. Initiation involves formation of the phagophore, a cup-shaped membrane structure in the cytoplasm. Nucleation involves the recruitment and assembly of several proteins including Beclin-1 and Vps34, among others [24]. Vps34 has a phosphoinositide 3-kinase (PI3K) activity and produces phosphatidylinositol 3-phosphate, which recruits molecular components involved in subse‐ quent vesicle elongation. Subsequently elongation is a critical step in forming the complete autophagosomes and is controlled by two ubiquitin-like conjugation systems. The first involves the Atg7 E1-like enzyme that mediates the covalent linkage of Atg12 to Atg5 followed by formation of a complex of Atg12-Atg5 with Atg16L, which dimerizes and associates with the phagophore membrane. In the second one LC3 has to be cleaved by Atg4 to generate the cytosolic LC3 (that arises from microtubular-associated protein-light chain) that can subse‐ quently be conjugated to a phosphatidylethanolamine tag by Atg7 and the E2-like enzyme Atg3. The resulting lipidated form of LC3, LC3-II, is attached to both sides of the autophago‐ some membrane. LC3-II is clearly necessary for autophagosome formation and is commonly used as a marker for autophagy. Finally, maturation and degradation steps, involves fusion with endosomes-lysosomes to form autophagic vacuoles (AVs) and requires a number of lysosomal proteins necessary for the degradation of the lumenal contents [25]. The transmem‐ brane Atg9 protein also contributes to autophagic vesicle nucleation and elongation, possibly by mediating the transport of lipid molecules. At this stage, LC3 on the outer autophagosome membrane is removed by Atg4 for reuse, while inner membrane LC3-II is digested along with the cargo. The rate-limiting step in the autophagic process is autophagosome formation.