bundles of actin filaments and have a role in cell migration, wound per ml cytoplasm). The filaments are formed by the ATP-dependent
healing and neurite growth. The protrusive thin and broad lamellipo- polymerization of actin monomer (with a molecular mass of 43 kDa)
dia, found at the leading edge of a motile cell, contain a branched into a characteristic string of beads in which the subunits are arranged
network of actin filaments. in a linear tight helix with a distance of 13 subunits between turns
The cytoskeleton restricts specific structures to particular cellular (Dominguez 2010). The polymerized filamentous form is termed
locations. For example, the Golgi apparatus is near the nucleus and F-actin (fibrillar actin) and the unpolymerized monomeric form is
endoplasmic reticulum, and mitochondria are near sites of energy known as G-actin (globular actin). Each monomer has an asymmetric
requirement. In addition, the cytoskeleton provides tracks for intracel- structure. When monomers polymerize, they confer a defined polarity
lular transport (e.g. shuttling vesicles and macromolecules, called on the filament: the plus or barbed end favours monomer addition,
cargoes, among cytoplasmic sites), the movement of chromosomes and the minus or pointed end favours monomer dissociation.
during cell division (mitosis and meiosis) or movement of the entire Treadmilling designates the simultaneous polymerization of an
cell during embryonic morphogenesis or the chemotactic extravascular actin filament at one end and depolymerization at the other end to
migration of leukocytes during homing. Examples of highly developed maintain its constant length.
and specialized functions of the cytoskeleton include the contraction See Bray (2001) for further reading.
of the sarcomere in striated muscle cells and the bending of the axoneme
of cilia and flagella. actin-binding proteins
The catalogue of cytoskeletal structural proteins is extensive and still A wide variety of actin-binding proteins are capable of modulating the
increasing. The major filamentous structures found in non-muscle cells form of actin within the cell. These interactions are fundamental to the
Monomer Tubulin dimer Tetramer Fig . 1 .8 Structural and molecular


features of cytoskeletal components .
G-actin–ATP β-tubulin GTP A, The actin filament (F-actin) is a
7 nm thick polymer chain of
α-tubulin GTP
ATP-bound G-actin monomers .
F-actin consists of a barbed (plus)
GTP
end, the initiation site of F-actin,
and a pointed (minus) end, the
Plus end dissociation site of F-actin . F-actin
can be severed and capped at the
Barbed end barbed end by gelsolin . B, The
microtubule is a 25 nm diameter
polymer of GTP-bound α-tubulin and
Unit length filament
GTP-bound β-tubulin dimers . The
dimer assembles at the plus end and
depolymerizes at the minus end . A
linear chain of α-tubulin/β-tubulin
dimers is called a protofilament . In
the end-on (top view), a microtubule
displays 13 concentrically arranged
7 nm thick 25 nm in diameter tubulin subunits . C, Tetrameric
complexes of intermediate filament
subunits associate laterally to form a
unit length filament consisting of
Intermediate filament
eight tetramers . Additional unit length
filaments anneal longitudinally and
generate a mature 10 nm thick
intermediate filament .
Severed actin filament Minus end
Gelsolin
Capped barbed end Protofilament
10 nm thick
Pointed end
Top view:
13 concentric tubulins
A Actin filament B Microtubule C Intermediate filament


Basic structure and function of cells
12.e1
1
RETPaHC
Septins are emerging as a novel cytoskeletal member because of their
filamentous organization and association with actin filaments and
microtubules. They are guanosine triphosphate (GTP)-binding proteins
that form hetero-oligomeric complexes (see Mostowy and Cossart
(2012) for additional information).
This polarity can be visualized in negatively stained images by allow-
ing F-actin to react with fragments containing the active head region of
myosin. Myosins bind to filamentous actin at an angle to give the
appearance of a series of arrowheads pointing towards the minus end
of the filament, with the barbs pointing towards the plus end.
It involves the addition of ATP-bound G-actin monomers at the
barbed end (fast-growing plus end) and removal of ADP-bound G-actin
at the pointed end (slow-growing minus end). Actin filaments grow or
shrink by addition or loss of G-actin monomer at both ends. Essentially,
actin polymerization in vitro proceeds in three steps: nucleation (aggre-
gation of G-actin monomers into a 3–4-monomer aggregate), elonga-
tion (addition of G-actin monomers to the aggregate) and a dynamic
steady state (treadmilling). Specific toxins (e.g. cytochalasins, phalloi-
dins and lantrunculins) bind to actin and affect its polymerization.
Cytochalasin D blocks the addition of new G-actin monomers to the
barbed end of F-actin; phalloidin binds to the interface between G-actin
monomers in F-actin, thus preventing depolymerization; and lantrun-
culin binds to G-actin monomers, blocking their addition to an actin
filament.


Cell structure
13
1
RETPaHC
Fig . 1 .9 The membrane at both ends), maintain a degree of active rigidity. Filamin
cytoskeleton . A, An interconnects adjacent actin filaments to produce loose filamentous
immunofluorescence gel-like networks composed of randomly orientated F-actin.
micrograph of α-actin F-actin can branch. The assembly of branched filamentous actin
microfilaments (green) in networks involves a complex of seven actin-related proteins 2/3
human airway smooth (Arp2/3) that is structurally similar to the barbed end of actin.
muscle cells in culture . See Rotty et al (2013) for further reading.
The actin-binding
Branched actin generated by the Arp2/3 protein complex localizes
protein, vinculin (red), is
at the leading edge of migrating cells, lamellipodia and phagosomes
localized at the ends of
(required for the capture by endocytosis and phagocytosis of particles
actin filament bundles;
and foreign pathogens by immune cells). Formin can elongate pre-
nuclei are blue . B, An
existing actin filaments by removing capping proteins at the barbed
immunofluorescence
end.
micrograph of keratin
intermediate filaments Other classes of actin-binding protein link the actin cytoskeleton to
(green) in human the plasma membrane either directly or indirectly through a variety of
keratinocytes in culture . membrane-associated proteins. The latter may also create links via
A
Desmosome junctions transmembrane proteins to the extracellular matrix. Best known of
are labelled with these is the family of spectrin-like molecules, which can bind to actin
antibody against and also to each other and to various membrane-associated proteins to
desmoplakin (red) . create supportive networks beneath the plasma membrane. Tetrameres
Nuclei are stained blue of spectrin α and β chains line the intracellular side of the plasma
(Hoechst) . C, An membrane of erythrocytes and maintain their integrity by their associa-
electron micrograph of tion with short actin filaments at either end of the tetramer.
human nerve showing Class V myosins are unconventional motor proteins transporting


microtubules (small, cargoes (such as vesicles and organelles) along actin filaments.
hollow structures in
Class I myosins are involved in membrane dynamics and actin organi-
cross-section, long
zation at the cell cortex, thus affecting cell migration, endocytosis,
arrow) in a transverse
pinocytosis and phagocytosis. Tropomyosin, an important regulatory
section of an
protein of muscle fibres, is also present in non-muscle cells, where
unmyelinated axon (A),
its function may be primarily to stabilize actin filaments against
engulfed by a Schwann
depolymerization.
cell (S) . Neuronal
intermediate filaments
Myosins, the motor proteins
(neurofilaments) are the
B solid, electron-dense The myosin family of microfilaments is often classified within a distinct
profiles, also in category of motor proteins. Myosin proteins have a globular head
transverse section (short region consisting of a heavy and a light chain. The heavy chain bears
arrow) . (A, Courtesy of an α-helical tail of varying length. The head has an ATPase activity and
Dr T Nguyen, Professor can bind to and move along actin filaments – the basis for myosin
J Ward, Dr SJ Hirst, function as a motor protein. The best-known class is myosin II, which
King’s College London . occurs in muscle and in many non-muscle cells. Its molecules have two
AA B, Courtesy of Prof . heads and two tails, intertwined to form a long rod. The rods can bind
Dr WW Franke, German to each other to form long, thick filaments, as seen in striated and
Cancer Research smooth muscle fibres and myoepithelial cells. Myosin II molecules can
Centre, Heidelberg . also assemble into smaller groups, especially dimers, which can cross-
C, Courtesy of Dr Bart
link individual actin microfilaments in stress fibres and other F-actin
Wagner, Histopathology
SS arrays. The ATP-dependent sliding of myosin on actin forms the basis
Department, Sheffield
for muscle contraction and the extension of microfilament bundles, as
Teaching Hospitals, UK .)
seen in cellular motility or in the contraction of the ring of actin and


myosin around the cleavage furrow of dividing cells. There are a number
of known subtypes of myosin II; they assemble in different ways and
have different dynamic properties. In skeletal muscle the myosin mol-
ecules form bipolar filaments 15 nm thick. Because these filaments have
C
a symmetric antiparallel arrangement of subunits, the midpoint is bare
of head regions. In smooth muscle the molecules form thicker, flattened
bundles and are orientated in random directions on either face of the
bundle. These arrangements have important consequences for the con-
organization of cytoplasm and to cell shape. The actin cytoskeleton is tractile force characteristics of the different types of muscle cell.
organized as closely packed parallel arrays of actin filaments forming Related molecules include the myosin I subfamily of single-headed
bundles or cables, or loosely packed criss-crossed actin filaments molecules with tails of varying length. Functions of myosin I include
forming networks (Fig. 1.9A). Actin-binding proteins hold together the movements of membranes in endocytosis, filopodial formation in
bundles and networks of actin filaments. Actin-binding proteins can neuronal growth cones, actin–actin sliding and attachment of actin to
be grouped into G-actin (monomer) binding proteins and F-actin membranes as seen in microvilli. As indicated above, molecular motors
(polymer) capping, cross-linking and severing proteins. Actin-binding of the myosin V family are implicated in the movements of cargoes on
proteins may have more than one function. actin filaments. So, for example, myosin Va transports vesicles along
Capping proteins bind to the ends of the actin filament either F-actin tracks in a similar manner to kinesin and cytoplasmic dynein-
to stabilize an actin filament or to promote its disassembly (see related cargo transport along microtubules. Each class of motor protein
Fig. 1.8). has different properties, but during cargo trafficking they often function
Cross-linking or bundling proteins tie actin filaments together in together in a coordinated fashion. (See Hammer 3rd and Sellers (2012)
longitudinal arrays to form bundles, cables or core structures. The for further reading on class V myosins.)
bundles may be closely packed in microvilli and filopodia, where paral-


lel filaments are tied tightly together to form stiff bundles orientated in Other thin filaments
the same direction. Cross-linking proteins of the microvillus actin A heterogeneous group of filamentous structures with diameters of
bundle core include fimbrin and villin. 2–4 nm occurs in various cells. The two most widely studied forms, titin
Other actin-bundling proteins form rather looser bundles of fila- and nebulin, constitute around 13% of the total protein of skeletal
ments that run antiparallel to each other with respect to their plus and muscle. They are amongst the largest known molecules and have
minus ends. They include myosin II, which can form cross-links with subunit weights of around 106; native molecules are about 1 µm in
ATP-dependent motor activity, and cause adjacent actin filaments to length. Their repetitive bead-like structure gives them elastic properties
slide on each other in the striated muscle sarcomere, and either change that are important for the effective functioning of muscle, and possibly
the shape of cells or (if the actin bundles are anchored into the cell for other cells.


Basic structure and function of cells
13.e1
1
RETPaHC
Profilin and thymosin β4 are G-actin binding proteins. Profilin binds In the presence of activated nucleation promotion factors, such as
to G-actin bound to ATP; it inhibits addition of G-actin to the slow- Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-
growing (pointed) end of F-actin but enables the fast-growing (barbed) homologous protein (WAVE, also known as SCAR), the Arp2/3 protein
end to grow faster and then dissociates from the actin filament. In addi- complex binds to the side of an existing actin filament (mother fila-
tion, profilin participates in the conversion of ADP back to the ATP–G- ment) and initiates the formation of a branching actin daughter fila-
actin bound form. Thymosin β4 binds to the ATP–G-actin bound form, ment at a 70° angle relative to the mother filament utilizing G-actin
preventing polymerization by sequestering ATP–G-actin into a reserve delivered to the Arp2/3 complex site.
pool. Spectrin-related molecules are present in many other cells. For
Members of the F-actin capping protein family are heterodimers instance, fodrin is found in neurones and dystrophin occurs in muscle
consisting of an α subunit (CPα) and a β subunit (CPβ) that cap the cells, linking the contractile apparatus with the extracellular matrix via
barbed end of actin filaments within all eukaryotic cells. Gelsolin has integral membrane proteins. Proteins such as ankyrin (which also binds
a dual role: it severs F-actin and caps the newly formed barbed end, actin directly), vinculin, talin, zyxin and paxillin connect actin-binding
blocking further filament elongation. proteins to integral plasma membrane proteins such as integrins
Fascin is an additional cross-linking protein. Villin is also a severing (directly or indirectly), and thence to focal adhesions (consisting of a
protein, causing the disassembly of actin filaments and the collapse of bundle of actin filaments attached to a portion of a plasma membrane


the microvillus. linked to the extracellular matrix).


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
14
1
NOITCES
Microtubules Fig . 1 .10 A duplicated
pair of centrioles in a
Microtubules are polymers of tubulin with the form of hollow, rela-
human carcinoma
tively rigid cylinders, approximately 25 nm in diameter and of varying
specimen . Each
length (up to 70 µm in spermatozoan flagella). They are present in most
TT centriole pair consists
cell types, being particularly abundant in neurones, leukocytes and
of a mother and
blood platelets. Microtubules are the predominant constituents of the
daughter, orientated
mitotic spindles of dividing cells and also form part of the axoneme of
approximately at right
cilia, flagella and centrioles. angles to each other so
Microtubules consist of tubulin dimers and microtubule-associated that one is sectioned
proteins. There are two major classes of tubulin: α- and β-tubulins. transversely (T) and the
Before microtubule assembly, tubulins are associated as dimers with a other longitudinally (L) .
combined molecular mass of 100 kDa (50 kDa each). Each protein The transversely
subunit is approximately 5 nm across and is arranged along the long sectioned centrioles
axis in straight rows of alternating α- and β-tubulins, forming protofila- are seen as rings of
ments (see Fig. 1.8). Typically, 13 protofilaments (the number can vary L microtubule triplets
between 11 and 16) associate in a ring to form the wall of a hollow (arrow) . (Courtesy of
cylindrical microtubule. Each longitudinal row is slightly out of align- Dr Bart Wagner,
ment with its neighbour, so that a spiral pattern of alternating α and β Histopathology
subunits appears when the microtubule is viewed from the side. There Department, Sheffield
Teaching Hospitals,
is a dynamic equilibrium between the dimers and assembled microtu-
UK .)
bules: dimeric asymmetry creates polarity (α-tubulins are all orientated
towards the minus end, β-tubulins towards the plus end). Tubulin is


added preferentially to the plus end; the minus end is relatively slow-
growing. Microtubules frequently grow and shrink at a rapid and con-
stant rate, a phenomenon known as dynamic instability, in which
growing tubules can undergo a ‘catastrophe’, abruptly shifting from net
growth to rapid shrinkage. The primary determinant of whether micro-
tubules grow or shrink is the rate of GTP hydrolysis. Tubulins are GTP-
binding proteins; microtubule growth is accompanied by hydrolysis of microtubules for considerable distances, thus enabling selective target-
GTP, which may regulate the dynamic behaviour of the tubules. Micro- ing of materials within the cell. Such movements occur in both direc-
tubule growth is initiated at specific sites, the microtubule-organizing tions along microtubules. Kinesin-dependent motion is usually towards
centres, of which the best known are centrosomes (from which most the plus ends of microtubules, e.g. from the cell body towards the axon
cellular microtubules polymerize) and the centriole-derived basal terminals in neurones, and away from the centrosome in other cells.
bodies (from which cilia grow). Microtubule-organizing centres include Conversely, dynein-related movements are in the opposite direction, i.e.
a specialized tubulin isoform known as γ-tubulin that is essential for to the minus ends of microtubules. Dyneins also form the arms of
the nucleation of microtubule growth. peripheral microtubules in cilia and flagella, where they make dynamic
Various drugs (e.g. colcemid, vinblastine, griseofulvin, nocodazole) cross-bridges to adjacent microtubule pairs. When these tethered
cause microtubule depolymerization by binding the soluble tubulin dyneins try to move, the resulting shearing forces cause the axonemal
dimers and so shifting the equilibrium towards the unpolymerized array of microtubules to bend, generating ciliary and flagellar beating
state. Microtubule disassembly causes a wide variety of effects, including movements. Kinesins form a large and diverse family of related


the inhibition of cell division by disruption of the mitotic spindle. microtubule-stimulated ATPases. Some kinesins are motors that move
Conversely, the drug paclitaxel (taxol) is a microtubule depolymeriza- cargo and others cause microtubule disassembly, whilst still others
tion inhibitor because it stabilizes microtubules and promotes abnor- cross-link mitotic spindle microtubules to push the two centriolar poles
mal microtubule assembly. Although this can cause a peripheral apart during mitotic prophase. See Bray (2001) for further reading.
neuropathy, paclitaxel is widely used as an effective chemotherapeutic
Centrioles, centrosomes and basal bodies
agent in the treatment of breast and ovarian cancer.
Centrioles are microtubular cylinders 0.2 µm in diameter and 0.4 µm
long (Fig. 1.10). They are formed by a ring of nine microtubule triplets
microtubule-associated proteins linked by a number of other proteins. At least two centrioles occur in
Various proteins that can bind to assembled tubulins may be concerned all animal cells that are capable of mitotic division (eggs, which undergo
with structural properties or associated with motility. One important meiosis instead of mitosis, lack centrioles). See Gönczy (2012) for
class of microtubule-associated proteins (MAPs) consists of proteins further reading on the structure and assembly of the centriole. They
that associate with the plus ends of microtubules. They regulate the usually lie close together, at right angles or, most usually, at an oblique
dynamic instability of microtubules as well as interactions with other angle to each other (an arrangement often termed a diplosome), within
cellular substructures. Structural MAPs form cross-bridges between adja- the centrosome, a densely filamentous region of cytoplasm at the centre
cent microtubules or between microtubules and other structures such of the cell. The centrosome is the major microtubule-organizing centre
as intermediate filaments, mitochondria and the plasma membrane. of most cells; it is the site at which new microtubules are formed and


Microtubule-associated proteins found in neurones include: MAPs 1A the mitotic spindle is generated during cell division. Centriole biogen-
and 1B, which are present in neuronal dendrites and axons; MAPs 2A esis is a complex process. At the beginning of the S phase (DNA replica-
and 2B, found chiefly in dendrites; and tau, found only in axons. MAP tion phase) of the cell cycle (see below), a new daughter centriole forms
4 is the major microtubule-associated protein in many other cell types. at right angles to each separated maternal centriole. Each mother–
Structural microtubule-associated proteins are implicated in microtu- daughter pair forms one pole of the next mitotic spindle, and the
bule formation, maintenance and disassembly, and are therefore of daughter centriole becomes fully mature only as the progeny cells are
considerable significance in cell morphogenesis, mitotic division, and about to enter the next mitosis. Because centrosomes are microtubule-
the maintenance and modulation of cell shape. Transport-associated organizing centres, they lie at the centre of a network of microtubules,
microtubule-associated proteins are found in situations in which move- all of which have their minus ends proximal to the centrosome.
ment occurs over the surfaces of microtubules, e.g. cargo transport, The microtubule-organizing centre contains complexes of γ-tubulin
bending of cilia and flagella, and some movements of mitotic spindles. that nucleate microtubule polymerization at the minus ends of micro-
They include a large family of motor proteins, the best known of which tubules. Basal bodies are microtubule-organizing centres that are closely
are the dyneins and kinesins. Another protein, dynamin, is involved in related to centrioles, and are believed to be derived from them. They
endocytosis. The kinetochore proteins assemble at the chromosomal are located at the bases of cilia and flagella, which they anchor to the
centromere during mitosis and meiosis. They attach (and thus fasten cell surface. The outer microtubule doublets of the axoneme of cilia and


chromosomes) to spindle microtubules; some of the kinetochore pro- flagella originate from two of the microtubules in each triplet of the
teins are responsible for chromosomal movements in mitotic and basal body.
meiotic anaphase.
All of these microtubule-associated proteins bind to microtubules microtubule-based transport of cargoes
and either actively slide along their surfaces or promote microtubule The transport of cargoes along microtubules via the motor proteins
assembly or disassembly. Kinesins and dyneins can simultaneously kinesin and cytoplasmic dynein respectively is the means by which
attach to membranes such as transport vesicles and convey them along neurotransmitters are delivered along axons to neuronal synapses


Basic structure and function of cells
14.e1
1
RETPaHC
The association of membrane vesicles with dynein motors means
that certain cytomembranes (including the Golgi apparatus) concen-
trate near the centrosome. This is convenient because the microtubules
provide a means of targeting Golgi vesicular products to different parts
of the cell.


Cell structure
15
1
RETPaHC
(anterograde axonal transport) and membrane-bound vesicles are sion. Of the different classes of intermediate filaments, keratin (cyto-
returned for recycling to the neuronal soma (retrograde axonal trans- keratin) proteins are found in epithelia, where keratin filaments are
port) (p. 45). In addition to anterograde and retrograde motor proteins, always composed of equal ratios of type I (acidic) and type II (basic to
the assembly and maintenance of all cilia and flagella involve the par- neutral) keratins to form heteropolymers. About 20 types of each of the
ticipation of non-membrane-bound macromolecular protein com- acidic and basic/neutral keratin proteins are known. For further reading
plexes called intraflagellar transport (IFT) particles. IFT particles localize on keratins in normal and diseased epithelia, see Pan et al (2012).
along the polarized microtubules of the axoneme, beneath the ciliary Within the epidermis, expression of keratin heteropolymers changes as
and flagellar membrane. IFT particles consist of two protein subcom- keratinocytes mature during their transition from basal to superficial
plexes: IFT-A (with a role in returning cargoes from the tip of the layers. Genetic abnormalities of keratins are known to affect the
axoneme to the cell body) and IFT-B (with a role in delivering cargoes mechanical stability of epithelia. For example, the disease epidermolysis
from the cell body to the tip of the axoneme). For further reading, see bullosa simplex is caused by lysis of epidermal basal cells and blistering
Scholey (2008) and Hao and Scholey (2009). of the skin after mechanical trauma. Defects in genes encoding keratins
During ciliogenesis, IFT requires the anterograde kinesin-2 motor 5 and 14 produce cytoskeletal instability leading to cellular fragility in
and the retrograde IFT-dynein motor to transport IFT particles–cargo the basal cells of the epidermis. When keratins 1 and 10 are affected,


complexes in opposite directions along the microtubules, from the cells in the spinous (prickle) cell layer of the epidermis lyse, and this
basal body to the tip of the ciliary axoneme and back again (intraciliary produces the intraepidermal blistering of epidermolytic hyperkeratosis.
transport). IFT is not just restricted to microtubules of cilia and flagella. See Porter and Lane (2003) for further reading.
During spermatid development, IFT particles–motor protein–cargo Type III intermediate filament proteins, including vimentin, desmin,
complexes appear to utilize microtubules of the manchette, a transient glial fibrillary acidic protein and peripherin, form homopolymer inter-
microtubule-containing structure, to deliver tubulin dimers and other mediate filaments. Vimentin is expressed in mesenchyme-derived cells
proteins by intramanchette transport during the development of the of connective tissue and some ectodermal cells during early develop-
spermatid tail (Kierszenbaum et al 2011). IFT also occurs along the ment; desmins in muscle cells; glial fibrillary acidic protein in glial
modified cilium of photoreceptor cells of the retina. Mutations in IFT cells; and peripherin in peripheral axons. Type IV intermediate fila-
proteins lead to the absence of cilia and are lethal during embryogen- ments include neurofilaments, nestin, syncoilin and α-internexin. Neu-
esis. Ciliopathies, many related to the defective sensory and/or mechan- rofilaments are a major cytoskeletal element in neurones, particularly
ical function of cilia, include retinal degeneration, polycystic kidney in axons (see Fig. 1.9C), where they are the dominant protein. Neuro-
disease, Bardet–Biedl syndrome, Jeune asphyxiating thoracic dystrophy, filaments (NF) are heteropolymers of low (NF–L), medium (NF–M)
respiratory disease and defective determination of the left–right axis. and high (NF–H) molecular weight (the NF–L form is always present


The seven-protein complex designated BBSome (for Bardet–Biedl syn- in combination with either NF–M or NF–H forms). Abnormal accumu-
drome, an obesity/retinopathy ciliopathy) is a component of the basal lations of neurofilaments (neurofibrillary tangles) are characteristic
body and participates in the formation of the primary cilium by regulat- features of a number of neuropathological conditions. Nestin resem-
ing the export and/or import of ciliary proteins. The transport of the bles a neurofilament protein, which forms intermediate filaments in
BBSome up and down and round about in cilia occurs in association neurectodermal stem cells in particular. The type V intermediate fila-
with anterograde IFT-B and retrograde IFT-A particles. For further ment group includes the nuclear lamins A, lamin B1 and lamin B2
reading on the BBSome, see Jin and Nachury (2009). For further reading lining the inner surface of the nuclear envelope of all nucleated cells.
on ciliogenesis, see Baldari and Rosenbaum (2010). Lamin C is a splice variant of lamin A. Lamins provide a mechanical
framework for the nucleus and act as attachment sites for a number of
Intermediate filaments
proteins that organize chromatin at the periphery of the nucleus. They
Intermediate filaments are about 10 nm thick and are formed by a are unusual in that they form an irregular anastomosing network of
heterogeneous group of filamentous proteins. In contrast to actin fila- filaments rather than linear bundles. See Burke and Stewart (2013) for
ments and microtubules, which are assembled from globular proteins further reading.
with nucleotide-binding and hydrolysing activity, intermediate fila-
ments consist of filamentous monomers lacking enzymatic activity.
Nucleus
Intermediate filament proteins assemble to form linear filaments in a
three-step process. First, a pair of intermediate filament protein sub-
units, each consisting of a central α-helical rod domain of about 310 The nucleus (see Figs 1.1–1.2) is generally the largest intracellular struc-
amino acids flanked by head and tail non-α-helical domains of varia- ture and is usually spherical or ellipsoid in shape, with a diameter of


ble size, form a parallel dimer through their central α-helical rod 3–10 µm. Conventional histological stains, such as haematoxylin or
domains coiled around each other. The variability of intermediate fila- toluidine blue, detect the acidic components (phosphate groups) of
ment protein subunits resides in the length and amino-acid sequence deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells and
of the head and tail domains, thought to be involved in regulating the tissue sections. DNA and RNA molecules are said to be basophilic
interaction of intermediate filaments with other proteins. Second, a because of the binding affinity of their negatively charged phosphate
tetrameric unit is formed by two antiparallel half-staggered coiled groups to basic dyes such as haematoxylin. A specific stain for DNA is
dimers. Third, eight tetramers associate laterally to form a 16 nm thick the Feulgen reaction.
unit length filament (ULF). Individual ULFs join end to end to form
Nuclear envelope
short filaments that continue growing longitudinally by annealing to
other ULFs and existing filaments. Filament elongation is followed by The nucleus is surrounded by the nuclear envelope, which consists of
internal compaction leading to the 30 nm thick intermediate filament an inner nuclear membrane (INM) and an outer nuclear membrane
(see Fig. 1.8). The tight association of dimers, tetramers and ULFs pro- (ONM), separated by a 40–50 nm perinuclear space that is spanned by
vides intermediate filaments with high tensile strength and resistance nuclear pore complexes (NPCs). The perinuclear space is continuous
to stretching, compression, twisting and bending forces. In contrast to with the lumen of the endoplasmic reticulum. The ONM has multiple
actin filaments and microtubules, intermediate filaments are non- connections with the endoplasmic reticulum, with which it shares its
polar (because of the antiparallel alignment of the initial tetramers) membrane protein components. The INM contains its own specific
and do not bind nucleot ides (as in G-actin and tubulin dimers), and integral membrane proteins (lamin B receptor and emerin, both pro-


ULFs anneal end to end to each other (in contrast to the polarized viding binding sites for chromatin bridging proteins). A mutation in
F-actin and microtubules, with one end, the plus end, growing faster the gene encoding emerin causes X-linked Emery–Dreifuss muscular
than the other end, the minus end). See Herrmann et al (2007) for dystrophy (EDMD), characterized by skeletal muscle wasting and
further reading. cardiomyopathy.
Intermediate filaments are found in different cell types and are often The nuclear lamina, a 15–20 nm thick, protein-dense meshwork, is
present in large numbers, either to provide structural strength where it associated with the inner face of the INM. The major components of
is needed (see Fig. 1.9B,C) or to provide scaffolding for the attachment the nuclear lamina are lamins, the type V intermediate filament proteins
of other structures. Intermediate filaments form extensive cytoplasmic consisting of A-type and B-type classes.
networks extending from cage-like perinuclear arrangements to the cell The nuclear lamina reinforces the nuclear membrane mechanically,
surface. Intermediate filaments of different molecular classes are char- determines the shape of the nucleus and provides a binding site for a
acteristic of particular tissues or states of maturity and are therefore range of proteins that anchor chromatin to the cytoskeleton. Nuclear
important indicators of the origins of cells or degrees of differentiation, lamin A, with over 350 mutations, is the most mutated protein linked
as well as being of considerable value in histopathology. to human disease. These are referred to as laminopathies, characterized
Intermediate filament proteins have been classified into five distinct by nuclear structural abnormalities that cause structurally weakened
types on the basis of their primary structure and tissue-specific expres- nuclei, leading to mechanical damage. Lamin A mutations cause a


Basic structure and function of cells
15.e1
1
RETPaHC
A-type lamins include lamin A (interacting with emerin), lamin C,
lamin C2 and lamin AΔ10 encoded by a single gene (LMNA). Lamin A
and lamin C are the major A-type lamins expressed in somatic cells,
whereas lamin C2 is expressed in testis. B-type lamins include lamin
B1 and lamin B2 (expressed in somatic cells), and testis-specific lamin
B3. Lamin B1 is encoded by the LMNB1 gene; lamin B2 is encoded by
the LMNB2 gene.


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
16
1
NOITCES
surprisingly wide range of diseases, from progeria to various dystro- permeable to small molecules, ions and proteins up to about 17 kDa.
phies, including an autosomal dominant form of EDMD. A truncated See Raices and D’Angelo (2012) for further reading on nuclear pore
farnesylated form of lamin A, referred to as progerin, leads to defects complex composition. Most proteins that enter the nucleus do so as
in cell proliferation and DNA damage of mesenchymal stem cells and complexes with specific transport receptor proteins known as import-
vascular smooth muscle cells. Affected patients display cardiovascular ins. Importins shuttle back and forth between the nucleus and cyto-
disease and die at an early age. Mice lacking lamin B1 and lamin B2 plasm. Binding of the cargo to the importin requires a short sequence
survive until birth; however, neuronal development is compromised of amino acids known as a nuclear localization sequence (NLS), and
when lamin B1 or lamin B2 is absent. Overexpression of lamin B1 is can either be direct or take place via an adapter protein. Interactions of
associated with autosomal dominant leukodystrophy characterized by the importin with components of the nuclear pore move it, together
gradual demyelination in the central nervous system. See Worman with its cargo, through the pore by an energy-independent process.
(2012) and Burke and Stewart (2013) for additional reading on lamins A complementary cycle functions in export of proteins and RNA mol-
and laminopathies. ecules from the nucleus to the cytoplasm using transport receptors
Condensed chromatin (heterochromatin) tends to aggregate near known as exportins.
the nuclear envelope during interphase. At the end of mitotic and A small GTPase called Ras-related nuclear protein (Ran) regulates the
meiotic prophase (see below), the lamin filaments disassemble by import and export of proteins across the nuclear envelope.
phosphorylation, causing the nuclear membranes to vesiculate and For further reading on the Ran pathway and exportins/importins, see
disperse into the endoplasmic reticulum. During the final stages of Clarke and Zhang (2008) and Raices and D’Angelo (2012).


mitosis (telophase), proteins of the nuclear periphery, including lamins,
Chromatin
associate with the surface of the chromosomes, providing docking sites
for membrane vesicles. Fusion of these vesicles reconstitutes the nuclear DNA is organized within the nucleus in a DNA–protein complex known
envelope, including the nuclear lamina, following lamin dephosphor- as chromatin. The protein constituents of chromatin are the histones
ylation. See Simon and Wilson (2011) for further reading on the and the non-histone proteins. Non-histone proteins are an extremely
nucleoskeleton. heterogeneous group that includes structural proteins, DNA and RNA
The transport of molecules between the nucleus and the cytoplasm polymerases, and gene regulatory proteins. Histones are the most abun-
occurs via specialized nuclear pore structures that perforate the nuclear dant group of proteins in chromatin, primarily responsible for the
membrane (Fig. 1.11A). They act as highly selective directional molecu- packaging of chromosomal DNA into its primary level of organization,
lar filters, permitting proteins such as histones and gene regulatory the nucleosome. There are four core histone proteins – H2A, H2B, H3
proteins (which are synthesized in the cytoplasm but function in the and H4 – which combine in equal ratios to form a compact octameric
nucleus) to enter the nucleus, and molecules that are synthesized in the nucleosome core. A fifth histone, H1, is involved in further compaction
nucleus but destined for the cytoplasm (e.g. ribosomal subunits, trans- of the chromatin. The DNA molecule (one per chromosome) winds
fer RNAs and messenger RNAs) to leave the nucleus. twice around each nucleosome core, taking up 165 nucleotide pairs.
Ultrastructurally, nuclear pores appear as disc-like structures with an This packaging organizes the DNA into a chromatin fibre 11 nm in
outer diameter of 130 nm and an inner pore with an effective diameter diameter, and imparts to this form of chromatin the electron micro-
for free diffusion of 9 nm (Fig. 1.11B). The nuclear envelope of an scopic appearance of beads on a string, in which each bead is separated


active cell contains up to 4000 such pores. The nuclear pore complex by a variable length of DNA, typically about 35 nucleotide pairs long.
has an octagonal symmetry and is formed by an assembly of more than The nucleosome core region and one of the linker regions constitute
50 proteins, the nucleoporins. The inner and outer nuclear membranes the nucleosome proper, which is typically about 200 nucleotide pairs
fuse around the pore complex (see Fig. 1.11A). Nuclear pores are freely in length. However, chromatin rarely exists in this simple form and is
usually packaged further into a 30 nm thick fibre, involving a single H1
histone per nucleosome, which interacts with both DNA and protein
to impose a higher order of nucleosome packing. Usually, 30 nm thick
N
fibres are further coiled or folded into larger domains. Individual
domains are believed to decondense and extend during active transcrip-
tion. In a typical interphase nucleus, euchromatin (nuclear regions that
appear pale in appropriately stained tissue sections, or relatively
electron-lucent in electron micrographs; see Fig. 1.2) is likely to consist
A mainly of 30 nm fibres and loops, and contains the transcriptionally
C
active genes. Transcriptionally active cells, such as most neurones, have
nuclei that are predominantly euchromatic. See Luger et al (2012) for
further reading on the nucleosome and chromatin structure.
Heterochromatin (nuclear regions that appear dark in appropriately
stained tissue sections or electron-dense in electron micrographs) is
characteristically located mainly around the periphery of the nucleus,
except over the nuclear pores (see Fig. 1.11A), and adjacent to the
nucleolus (see Fig. 1.2). It is a relatively compacted form of chromatin
in which the histone proteins carry a specific set of post-translational
modifications, including methylation at characteristic residues. This
facilitates the binding of specific heterochromatin-associated proteins.
Heterochromatin includes non-coding regions of DNA, such as centro-
meric regions, which are known as constitutive heterochromatin. DNA
becomes transcriptionally inactive in some cells as they differentiate
during development or cell maturation, and contributes to heterochro-


matin; it is known as facultative heterochromatin. The inactive X chro-
mosome in females is an example of facultative heterochromatin and
can be identified in the light microscope as the deeply staining Barr
B
body often located near the nuclear periphery or a drumstick extension
of a nuclear lobe of a mature multilobed neutrophil leukocyte.
Fig . 1 .11 A, The nuclear envelope with nuclear pores (arrows) in
In transcriptionally inactive cells, chromatin is predominantly in the
transverse section, showing the continuity between the inner and outer
condensed, heterochromatic state, and may comprise as much as 90%
phospholipid layers of the envelope on either side of the pore . The fine
of the total. Examples of such cells are mature neutrophil leukocytes
‘membrane’ appearing to span the pore is formed by proteins of the pore
(in which the condensed chromatin is present in a multilobular, densely
complex . Note that the chromatin is less condensed in the region of
staining nucleus) and the highly condensed nuclei of orthochromatic
nuclear pores . Abbreviations: N, nucleus; C, cytoplasm . B, Nuclear pores
erythroblasts (late-stage erythrocyte precursors). In most mature cells,
seen ‘en face’ as spherical structures (arrows) in a tangential section
through the nuclear envelope . The appearance of the envelope varies in a mixture of the two occurs, indicating that only a proportion of the
electron density as the plane of section passes through different regions DNA is being transcribed. A particular instance of this is seen in the B
of the curved double membrane, which is interrupted at intervals by pores lymphocyte-derived plasma cell, in which much of the chromatin is in
through the envelope (see also Fig . 1 .1) . The surrounding cytoplasm with the condensed condition and is arranged in regular masses around the
ribosomes is less electron-dense . Human tissues . (Courtesy of Dr Bart perimeter of the nucleus, producing the so-called ‘clock-face’ nucleus
Wagner, Histopathology Department, Sheffield Teaching Hospitals, UK .) (see Figs 4.6, 4.12). Although this cell is actively transcribing, much of


Cell structure
17
1
RETPaHC
its protein synthesis is of a single immunoglobulin type, and conse- easily seen during metaphase, although prophase chromosomes can be
quently much of its genome is in an inactive state. used for more detailed analyses.
During mitosis, the chromatin is further reorganized and condensed Lymphocytes separated from blood samples, or cells taken from
to form the much-shortened chromosomes characteristic of metaphase. other tissues, are used as a source of chromosomes. Diagnosis of fetal
This shortening is achieved through further levels of close packing of chromosome patterns is generally carried out on samples of amniotic
the chromatin. The condensed chromosomes are stabilized by protein fluid containing fetal cells aspirated from the uterus by amniocentesis,
complexes known as condensins. Progressive folding of the chromo- or on a small piece of chorionic villus tissue removed from the placenta.
somal DNA by interactions with specific proteins can reduce 5 cm of Whatever their origin, the cells are cultured in vitro and stimulated to
chromosomal DNA by 10,000-fold, to a length of 5 µm in the mitotic divide by treatment with agents that stimulate cell division. Mitosis is
chromosome. interrupted at metaphase with spindle inhibitors. The chromosomes are
dispersed by first causing the cells to swell in a hypotonic solution, then
Chromosomes and telomeres
the cells are gently fixed and mechanically ruptured on a slide to spread
The nuclear DNA of eukaryotic cells is organized into linear units called the chromosomes. They are subsequently stained in various ways to
chromosomes. The DNA in a normal human diploid cell contains allow the identification of individual chromosomes by size, shape and
6 × 109 nucleotide pairs organized in the form of 46 chromosomes (44 distribution of stain (Fig. 1.12). General techniques show the obvious
autosomes and 2 sex chromosomes). The largest human chromosome landmarks, e.g. lengths of arms and positions of constrictions. Banding
(number 1) contains 2.5 × 108 nucleotide pairs, and the smallest (the Y techniques demonstrate differential staining patterns, characteristic for
chromosome) 5 × 107 nucleotide pairs. each chromosome type. Fluorescence staining with quinacrine mustard
Each chromosomal DNA molecule contains a number of specialized and related compounds produces Q bands, and Giemsa staining (after


nucleotide sequences that are associated with its maintenance. One is treatment that partially denatures the chromatin) gives G bands (Fig.
the centromeric DNA region. During mitosis, a disc-shaped structure 1.12A). Other less widely used methods include: reverse Giemsa stain-
composed of a complex array of proteins, the kinetochore, forms as a ing, in which the light and dark areas are reversed (R bands); the stain-
substructure at the centromeric region of DNA to which kinetochore ing of constitutive heterochromatin with silver salts (C-banding); and
microtubules of the spindle attach. Another region, the telomere, T-banding to stain the ends (telomeres) of chromosomes. Collectively,
defines the end of each chromosomal DNA molecule. Telomeres consist these methods permit the classification of chromosomes into num-
of hundreds of repeats of the nucleotide sequence (TTAGGG) n. The very bered autosomal pairs in order of decreasing size, from 1 to 22, plus
ends of the chromosomes cannot be replicated by the same DNA the sex chromosomes.
polymerase as the rest of the chromosome, and are maintained by a A summary of the major classes of chromosome is given in
specific enzyme called telomerase, which contains an RNA subunit Table 1.1.
acting as the template for lengthening the TTAGGG repeats. See Methodological advances in banding techniques improved the re-
Nandakumar and Cech (2013) for further reading on the recruitment cognition of abnormal chromosome patterns. The use of in situ hybridi-
of telomerase to telomeres. Thus telomerase is a specialized type of zation with fluorescent DNA probes specific for each chromosome (Fig.
polymerase known as a reverse transcriptase that turns sequences in 1.12B) permits the identification of even very small abnormalities.
RNA back into DNA. The number of tandem repeats of the telomeric
Nucleolus
DNA sequence varies. The telomere appears to shorten with successive
cell divisions because telomerase activity reduces or is absent in dif- Nucleoli are a prominent feature of an interphase nucleus (see Fig. 1.2).
ferentiated cells with a finite lifespan. In mammals, telomerase is active They are the site of most of the synthesis of ribosomal RNA (rRNA) and


in the germ-cell lineage and in stem cells, but its expression in somatic assembly of ribosome subunits. Nucleoli organize at the end of mitosis
cells may lead to or prompt cancer. A lack of telomere maintenance
determines the shrinking of telomeres in proliferating cells to the point
when cells stop dividing, a condition known as replicative senescence. Table 1.1 Summary of the major classes of chromosome
See Sahin and DePinho (2012) for further reading on telomeres and
Group Features
progressive DNA damage.
The role of the telomere in ageing and cell senescence is further 1–3 (A) Large metacentric chromosomes
discussed at the end of this chapter. 4–5 (B) Large submetacentric chromosomes
6–12 + X (C) Metacentrics of medium size
Karyotypes: classification of human chromosomes
13–15 (D) Medium-sized acrocentrics with satellites
A number of genetic abnormalities can be directly related to the chro- 16–18 (E) Shorter metacentrics (16) or submetacentrics (17,18)
mosomal pattern. The characterization or karyotyping of chromosome
19–20 (F) Shortest metacentrics
number and structure is therefore of considerable diagnostic impor-
21–22 + Y (G) Short acrocentrics; 21, 22 with satellites, Y without
tance. The identifying features of individual chromosomes are most
1 2 3 4 5
1 2 3 4 5
6 7 8 9 10 11 12 6 7 8 9 10 11 12
13 14 15 16 17 18 13 14 15 16 17 18
A 19 20 21 22 X Y B 19 20 21 22 X Y
Fig . 1 .12 Chromosomes from normal males, arranged as karyotypes . A, G-banded preparation . B, Preparation stained by multiplex fluorescence in situ
hybridization to identify each chromosome . (Courtesy of Dr Denise Sheer, Cancer Research UK .)


Basic structure and function of cells
17.e1
1
RETPaHC
Telomerase has been associated with ageing and cell senescence
because a gradual loss of telomeres may lead to tissue atrophy, stem
cell depletion and deficient tissue repair or regeneration. Mutations
causing loss of function of telomerase or the RNA-containing template
have been associated with dyskeratosis congenita (characterized by
abnormal skin pigmentation, nail dystrophy and mucosal leukoplasia),
aplastic anaemia and pulmonary fibrosis.


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
18
1
NOITCES
and consist of repeated clusters of ribosomal DNA (rDNA) genes and Cdk1
processing molecules responsible for producing ribosome subunits. The
initial step of the assembly of a ribosome subunit starts with the tran- G2 Cyclin A
scription of rDNA genes by RNA polymerase I. The rDNA genes,
arranged in tandem repeats called nucleolar organizing regions (NORs), Cdk1
are located on acrocentric chromosomes. There are five pairs of acro- Mitosis
centric chromosomes in humans. The initial 47S rRNA precursor tran- Cyclin B
script is cleaved to form the mature 28S, 18S and 5.8S rRNAs, assembled
Checkpoint 2
with the 5S rRNA (synthesized by RNA polymerase III outside the
nucleolus) and coupled to small nucleolar ribonucleoproteins and
other non-ribosomal proteins to form 60S (containing 28S rRNA, 5.8S
rRNA and 5S rRNA) and 40S (containing 18S rRNA) preribosome sub-
units. These are then exported to the cytoplasm across nuclear pores as S
mature ribosome subunits. About 726 human nucleolar proteins have
Checkpoint 1 Cyclin D
been identified by protein purification and mass spectrometry. For
further reading on nucleolar functions, see Boisvert et al (2007).
Cdk2 Cdk4
Ribosomal biogenesis occurs in distinct subregions of the nucleolus, G1
visualized by electron microscopy. The three nucleolar subregions are Cyclin A
fibrillar centres (FCs), dense fibrillar components (DFCs) and granular
components (GCs). Transcription of the rDNA repeats takes place at
the FC-DFC boundary; pools of RNA polymerase I reside in the FC
Cyclin E
region; processing of transcripts and coupling to small nucleolar ribo-
nucleoproteins take place in DFC; and the assembly of ribosome sub- Cdk2
units is completed in the GC region. Fig . 1 .13 The cell cycle consists of an interphase (G phase, S phase and
1


The nucleolus is disassembled when cells enter mitosis and tran- G phase) followed by mitosis . The cyclin D/Cdk4 complex assembles at
2
scription becomes inactive. It reforms after nuclear envelope reorganiza- the beginning of G; the cyclin E/Cdk2 complex assembles near the end
1
tion in telophase, in a process associated with the onset of transcription of G as the cell is preparing to cross checkpoint 1 to start DNA synthesis
1
in nucleolar organizing centres on each specific chromosome, and (during S phase) . The cyclin A/Cdk2 complex assembles as DNA
becomes functional during the G phase of the cell cycle. An adequate synthesis starts . Completion of G is indicated by the assembled cyclin A/
1 2
pool of ribosome subunits during cell growth and cell division requires Cdk1 complex . A cell crosses checkpoint 2 to initiate mitosis when the
steady nucleolar activity to support protein synthesis. Several DNA cyclin B/Cdk1 complex assembles . The cyclin B/Cdk1 complex is
helicases, a conserved group of enzymes that unwind DNA, accumulate degraded by the 26S proteasome and an assembled cyclin D/Cdk4 marks
in the nucleolus under specific conditions such as Bloom’s syndrome the start of the G 1 phase of a new cell cycle . For details, see text .
(an autosomal recessive disorder characterized by growth deficiency, (Modified with permission from Kierszenbaum AL, Tres LL . Histology and
Cell Biology: An Introduction to Pathology . 3rd ed, Philadelphia: Elsevier,
immunodeficiency and a predisposition to cancer) and Werner’s syn-
Saunders; 2011 .)
drome (an autosomal recessive condition characterized by the early
appearance of various age-related diseases).
certain tumour suppressor genes (e.g. the gene mutated in retinoblas-
toma, Rb) block the cycle in G. DNA synthesis (replication of the
CELL DIVISION AND THE CELL CYCLE 1
genome) occurs during S phase, at the end of which the DNA content
of the cell has doubled. During G, the cell prepares for division; this
2
During prenatal development, most cells undergo repeated division period ends with the onset of chromosome condensation and break-


(see Video 1.1) as the body grows in size and complexity. As cells down of the nuclear envelope. The times taken for S, G and M are
2
mature, they differentiate structurally and functionally. Some cells, such similar for most cell types, and occupy 6–8, 2–4 and 1–2 hours respec-
as neurones, lose the ability to divide. Others may persist throughout tively. In contrast, the duration of G shows considerable variation,
1
the lifetime of the individual as replication-competent stem cells, e.g. sometimes ranging from less than 2 hours in rapidly dividing cells to
cells in the haemopoietic tissue of bone marrow. Many stem cells divide more than 100 hours, within the same tissue.
infrequently, but give rise to daughter cells that undergo repeated cycles The passage of a cell through the cell cycle is controlled by proteins
of mitotic division as transit (or transient) amplifying cells. Their divi- in the cytoplasm: cyclins and cyclin-dependent kinases (Cdks; Fig
sions may occur in rapid succession, as in cell lineages with a short 1.13). Cyclins include G cyclins (D cyclins), S-phase cyclins (cyclins E
1
lifespan and similarly fast turnover and replacement time. Transit and A) and mitotic cyclins (B cyclins). Cdks, protein kinases, which are
amplifying cells are all destined to differentiate and ultimately to die activated by binding of a cyclin subunit, include G Cdk (Cdk4), an
1
and be replaced, unlike the population of parental stem cells, which S-phase Cdk (Cdk2) and an M-phase Cdk (Cdk1). Cell cycle progres-
self-renews. sion is driven in part by changes in the activity of Cdks. Each cell cycle
Patterns and rates of cell division within tissues vary considerably. stage is characterized by the activity of one or more Cdk–cyclin pairs.
In many epithelia, such as the crypts between intestinal villi, the replace- Transitions between cell cycle stages are triggered by highly specific
ment of damaged or ageing cells by division of stem cells can be rapid. proteolysis by the 26S proteasome of the cyclins and other key


Rates of cell division may also vary according to demand, as occurs in components.
the healing of wounded skin, in which cell proliferation increases to a To give one example, the transition from G to mitosis is driven by
2
peak and then returns to the normal replacement level. The rate of cell activation of Cdk1 by its partners, the A- and B-type cyclins; the char-
division is tightly coupled to the demand for growth and replacement. acteristic changes in cellular structure that occur as cells enter mitosis
Where this coupling is faulty, tissues either fail to grow or replace their are largely driven by phosphorylation of proteins by active Cdk1-cyclin
cells, or they can overgrow, producing neoplasms. A and Cdk1-cyclin B. Cells exit from mitosis when an E3 ubiquitin
The cell cycle is an ordered sequence of events, culminating in cell ligase, the anaphase promoting complex, also called cyclosome
growth and division to produce two daughter cells. It generally lasts a (APC/C), marks the cyclins for destruction. In addition, APC/C prompts
minimum of 12 hours, but in most adult tissues can be considerably the degradation of the mitotic cyclin B and the destruction of cohesins,
longer, and is divided into four distinct phases, which are known as G thus allowing sister chromatids to separate.
1
(for gap 1), S (for DNA synthesis), G (for gap 2) and M (for mitosis). There are important checkpoints in the cell cycle (see Fig. 1.13).
2
The combination of G, S and G phases is known as interphase. M is Checkpoint 1 requires G cyclins to bind to their corresponding Cdks
1 2 1
the mitotic phase, which is further divided into four phases (see below). to signal the cell to prepare for DNA synthesis. S-phase promoting
G is the period when cells respond to growth factors directing the cell factor (SPF; cyclin A bound to Cdk2) enters the nucleus to stimulate
1
to initiate another cycle; once made, this decision is irreversible. It is DNA synthesis. Checkpoint 2 requires M-phase promoting factor
also the phase in which most of the molecular machinery required to (mitotic cyclin B bound to M-phase Cdk1) to trigger the assembly of


complete another cell cycle is generated. Centrosomes duplicate during the mitotic spindle, breakdown of the nuclear envelope, arrest of gene
S phase in preparation for mitosis. Cells that retain the capacity for transcription and condensation of chromosomes. During metaphase of
proliferation, but which are no longer dividing, have entered a phase mitosis, M-phase promoting factor activates APC/C, which determines
called G and are described as quiescent even though they may be quite the breakdown of cohesins, the protein complex holding sister chroma-
0
active physiologically. Growth factors can stimulate quiescent cells to tids together. Then, at anaphase, separated chromatids move to the
leave G and re-enter the cell cycle, whereas the proteins encoded by opposite poles of the spindle. Finally, B cyclins are destroyed following
0


Basic structure and function of cells
18.e1
1
RETPaHC
The targets for proteolysis are marked for destruction by E3 ubiquitin
ligases, which decorate them with polymers of the small protein ubiq-
uitin, a sign for recognition by the 26S proteasome.


Cell division and the cell cycle
19
1
RETPaHC
their attachment to ubiquitin, targeting them for destruction by the 26S Prophase
proteasome. As G starts, cyclins D, bound to Cdk4, start preparation
1 Nuclear
for a new cell cycle. membrane Centriole centre
Quality control checkpoint 2 operates to delay cell-cycle progression of aster (or spindle pole)
Centromere
when DNA has been damaged by radiation or chemical mutagens. Cells
with checkpoint defects, such as loss of the protein p53, which is a Microtubules
Two sister of spindle
major negative control element in the division cycle of all cells, are
chromatids
commonly associated with the development of malignancy. An example attached at
is Li Fraumeni syndrome, where a defective p53 gene leads to a high centromere
frequency of cancer in affected individuals. In cells, p53 protein binds
DNA and stimulates another gene to produce p21 protein, which inter-
acts with Cdk2 to prevent S-phase promoting activity. When mutant
p53 can no longer bind DNA to stimulate production of p21 to stop Prometaphase
DNA synthesis, cells acquire oncogenic properties. The p53 gene is an
example of a tumour suppressor gene. For further reading on p53 muta- Spindle pole
tions and cancer, see Muller and Vousden (2013).
Nuclear
Mitosis and meiosis membrane Microtubule
vesicles
Mitosis is the process that results in the distribution of identical copies
of the parent cell genome to the two daughter somatic cells. In meiosis,
the divisions immediately before the final production of gametes halve
the number of chromosomes to the haploid number, so that at fertiliza- Metaphase
tion the diploid number is restored. Moreover, meiosis includes a phase Cell equator
in which exchange of genetic material occurs between homologous
chromosomes. This allows a rearrangement of genes to take place,
which means that the daughter cells differ from the parental cell in both
their precise genetic sequence and their haploid state. Mitosis and
meiosis are alike in many respects, and differ principally in chromo-
somal behaviour during the early stages of cell division. In meiosis, two
divisions occur in succession, without an intervening S phase. Meiosis


I is distinct from mitosis, whereas meiosis II is more like mitosis.
Mitosis
New DNA is synthesized during the S phase of the cell cycle interphase. Anaphase
This means that the amount of DNA in diploid cells has doubled to
the tetraploid value by the onset of mitosis, although the chromosome
number is still diploid. During mitosis, this amount is halved between
the two daughter cells, so that DNA quantity and chromosome number Chromatids pulled
are diploid in both cells. The cellular changes that achieve this distribu- toward pole of spindle
as their microtubules
tion are conventionally divided into four phases called prophase, meta- shorten
phase, anaphase and telophase (Figs 1.14–1.15, Video 1.1).
Prophase
During prophase, the strands of chromatin, which are highly extended
during interphase, shorten, thicken and resolve themselves into recog-
nizable chromosomes. Each chromosome is made up of duplicate chro- Telophase
matids (the products of DNA replication) joined at their centromeres. Chromosomes decondense and
Outside the nucleus, the two centriole pairs begin to separate, and move detach from microtubules
towards opposite poles of the cell. Parallel microtubules are assembled
between them to create the mitotic spindle, and others radiate to form
the microtubule asters, which come to form the spindle poles or mitotic
centre. As prophase proceeds, the nucleoli disappear, and the nuclear
envelope suddenly disintegrates to release the chromosomes, an event
that marks the end of prophase. Nuclear membrane
reforms
Prometaphase–metaphase
As the nuclear envelope disappears, the spindle microtubules extend
into the central region of the cell, attaching to the chromosomes, which Cytokinesis
subsequently move towards the equator of the spindle (prometaphase).
The spindle consists of kinetochore microtubules attached to the kine-
tochore, a multiprotein structure assembled at the centromeric DNA
region, and polar microtubules, which are not attached to chromo- Centriole
somes but instead overlap with each other at the centre of the cell. The
grouping of chromosomes at the spindle equator is called the meta-


phase or equatorial plate. The chromosomes, attached at their centro-
Nuclear
meres, appear to be arranged in a ring when viewed from either pole membrane
of the cell, or to lie linearly across this plane when viewed from above.
Cytoplasmic movements during late metaphase effect the approxi-
mately equal distribution of mitochondria and other cell structures Actin–myosin belt
around the cell periphery.
Fig . 1 .14 The stages in mitosis, including the appearance and distribution
of the chromosomes .
anaphase
By the end of metaphase every chromosome consists of a pair of sister
chromatids attached to opposing spindle poles by bundles of microtu- microtubule-dependent pulling forces. Proteolytic cleavage releases the
bules associated with the kinetochore. The onset of anaphase begins cohesion between sister chromatids, which then move towards opposite
with the proteolytic cleavage by the enzyme separase of a key subunit spindle poles while the microtubule bundles attached to the kineto-
of protein complexes known as cohesins. The latter hold the replicated chores shorten and move polewards. At the end of anaphase the sister
sister chromatids together to resist separation even when exposed to chromatids are grouped at either end of the cell, and both clusters are


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
20
1
NOITCES
Fig . 1 .15 diploid in number. An infolding of the cell equator begins, deepening
Immunofluorescence during telophase as the cleavage furrow.
images of stages in
mitosis in human Telophase
carcinoma cells in During telophase the nuclear envelopes reform, beginning with the
culture . A, Metaphase, association of membranous vesicles with the surface of the chromo-
with spindle somes. Later, after the vesicles have fused and the nuclear envelope is
microtubules (green), complete, the chromosomes decondense and the nucleoli reform. At
the microtubule-
the same time, cytoplasmic division, which usually begins in early
stabilizing protein
A anaphase, continues until the new cells separate, each with its derived
(HURP; red) and
nucleus. The spindle remnant now disintegrates. While the cleavage
chromosomal DNA
furrow is active, a peripheral band or belt of actin and myosin appears
(blue) . B, Anaphase,
in the constricting zone; contraction of this band is responsible for
with spindle
furrow formation.
microtubules (green),
the central spindle Failure of disjunction of chromatids, so that sister chromatids pass
(Aurora-B kinase, red) to the same pole, may sometimes occur. Of the two new cells, one will
and segregated have more, and the other fewer, chromosomes than the diploid number.
chromosomes (blue) . Exposure to ionizing radiation promotes non-disjunction and may, by
C, Late anaphase, with chromosomal damage, inhibit mitosis altogether. A typical symptom of
spindle microtubules radiation exposure is the failure of rapidly dividing epithelia to replace
B (green), the central lost cells, with consequent ulceration of the skin and mucous mem-
spindle (Plk1 kinase, branes. Mitosis can also be disrupted by chemical agents, particularly
red, appearing yellow vinblastine, paclitaxel (taxol) and their derivatives. These compounds
where co-localized with either disassemble spindle microtubules or interfere with their dynam-


microtubule protein) ics, so that mitosis is arrested in metaphase.
and segregated
chromosomes (blue) . Meiosis
(Courtesy of Dr Herman
There are two consecutive cell divisions during meiosis: meiosis I and
Silljé, Max-Planck-
meiosis II (Fig. 1.16). Details of this process differ at a cellular level for
Institut für Biochemie,
male and female lineages.
Martinsried, Germany .)
C
A Events preceding meiosis
Centromere Paired sister
centromeres
Premeiotic Meiotic
S phase prophase
B Meiotic prophase
A a A
b a
B paP ta ei rr nin ag l ao nf d B b Meiosis I
maternal
homologues
Leptotene Zygotene Pachytene Diplotene Diakinesis
C Meiosis I
A
A B Chiasmata A B B
b
a b a b Meiosis I a Meiosis II
Interphase
Metaphase I Anaphase I (no S phase)
D Meiosis II
A
B
a
b
Prophase II Metaphase II Anaphase II Haploid gametes
Fig . 1 .16 The stages in meiosis, depicted by two pairs of maternal and paternal homologues (dark and pale colours) . DNA and chromosome
complement changes and exchange of genetic information between homologues are indicated .


Cell polarity and domains
21
1
RETPaHC
meiosis I equatorial plane of the spindle. The centromeres of each pair of sister
Prophase I chromatids function as a single unit, facing a single spindle pole.
Meiotic prophase I is a long and complex phase that differs consider- Homologous chromosomes are pulled towards opposite spindle poles,
ably from mitotic prophase and is customarily divided into five sub- but are held paired at the spindle midzone by chiasmata. Errors in
stages, called leptotene, zygotene, pachytene, diplotene and diakinesis. chromosome segregation (known as non-disjunction) lead to the pro-
There are three distinctive features of male meiotic prophase that are duction of aneuploid progeny. Most human aneuploid embryos are
not seen during mitotic prophase: the pairing, or synapse, of homolo- non-viable and this is the major cause of fetal loss (spontaneous abor-
gous chromosomes of paternal and maternal origin to form bivalent tion), particularly during the first trimester of pregnancy in humans.
structures; the organization of nucleoli by autosomal bivalents; and The most common form of viable aneuploid progeny in humans is
significant non-ribosomal RNA synthesis by autosomal bivalents (in Down’s syndrome (trisomy for chromosome 21), which exhibits a dra-
contrast to the transcriptional inactivity of the XY chromosomal pair) matic increase with maternal age.
(see Tres 2005). In the female, meiotic prophase I starts during fetal
Anaphase and telophase I
gonadogenesis, is arrested at the diplotene stage and resumes at puberty.
In the male, meiosis starts at puberty. Anaphase I of meiosis begins with the release of cohesion between the
arms of sister chromatids, much as it does during mitosis. As position-
Leptotene stage During leptotene, homologous chromosomes ing of bivalent pairs is random, assortment of maternal and paternal
(maternal and paternal copies of the same chromosome), replicated in chromosomes in each telophase nucleus is also random. Critically,
a preceding S phase and each consisting of sister chromatids joined at sister centromeres, and thus chromatids, do not separate during ana-


the centromere (see above), locate one another within the nucleus, and phase I.
the process of genetic recombination is initiated. Cytologically, chro- During meiosis I, cytoplasmic division occurs by specialized mecha-
mosomes begin to condense, appearing as individual threads that are nisms. In females, the division is highly asymmetric, producing one egg
attached via their telomeres to the nuclear envelope. They often show and one tiny cell known as a polar body. In males, the process results
characteristic beading throughout their length. in production of spermatocytes that remain joined by small cytoplas-
mic bridges.
Zygotene stage During zygotene, the homologous chromosomes
meiosis II
initiate pairing or synapsis, during which they become intimately asso-
ciated with one another. Synapsis may begin near the telomeres at the Meiosis II commences after only a short interval during which no DNA
inner surface of the nuclear membrane, and during this stage the tel- synthesis occurs. The centromeres of sister chromatids remain paired,
omeres often cluster to one side of the nucleus (a stage known as the but rotate so that each one can face an opposite spindle pole. Onset of
bouquet because the chromosomes resemble a bouquet of flowers). The anaphase II is triggered by loss of cohesion between the centromeres,
pairs of synapsed homologues, also known as bivalents, are linked as it is in mitosis. This second division is more like mitosis, in that
together by a tripartite ribbon, the synaptonemal complex, which con- chromatids separate during anaphase, but, unlike mitosis, the separat-
sists of two lateral dense elements and a central, less dense, linear ing chromatids are genetically different (the result of genetic recombi-
element. nation). Cytoplasmic division also occurs and thus, in the male, four
The sex chromosomes also start to synapse during zygotene. In haploid cells, interconnected by cytoplasmic bridges, result from
males, with distinct X and Y chromosomes, synapsis involves a region meiosis I and II.
of shared DNA sequence known as the pseudoautosomal region. The
XY bivalent adopts a special condensed structure, known as the sex
CELL POLARITY AND DOMAINS


vesicle, which becomes associated later at pachytene with migratory
nucleolar masses originating in the autosomal bivalents.
Chromosome behaviour in meiosis is intimately linked with the Epithelia are organized into sheets or glandular structures with very
process of genetic recombination. This begins during leptotene, as different environments on either side. These cells actively transfer mac-
homologous chromosomes first locate one another at a distance. Syn- romolecules and ions between the two surfaces and are thus polarized
apsis, stabilized by the synaptonemal complex, facilitates recombina- in structure and function. In polarized cells, particularly in epithelia,
tion, as sites of genetic exchange are turned into specialized structures the cell is generally subdivided into domains that reflect the polariza-
known as chiasmata, which are topological crossing-over points that tion of activities within it. The free surface, e.g. that facing the intestinal
hold homologous chromosomes together. lumen or airway, is the apical surface, and its adjacent cytoplasm is the
apical cell domain. This is where the cell interfaces with a specific body
Pachytene stage When synapsis is complete for all chromosomes, compartment (or, in the case of the epidermis, with the outside world).
the cell is said to be in pachytene. Each bivalent looks like a single thick The apical surface is specialized to act as a barrier, restricting access of
structure, but is actually two pairs of sister chromatids held together by substances from this compartment to the rest of the body. Specific
the synaptonemal complex. Genetic recombination between non-sister components are selectively absorbed from, or added to, the external
chromatids is completed at this point, with sites where it has occurred compartment by the active processes, respectively, of active transport
(usually one per chromosome arm) appearing as recombination and endocytosis inwardly or exocytosis and secretion outwardly.
nodules in the centre of the synaptonemal complex. The apical surface is often covered with small protrusions of the cell
surface, microvilli, which increase the surface area, particularly for
Diplotene stage During diplotene, the synaptonemal complex disas- absorption.
sembles and pairs of homologous chromosomes, now much shortened, The surface of the cell opposite to the apical surface is the basal


separate, except where crossing over has occurred (chiasmata). This surface, with its associated basolateral cell domain. In a single-layered
process is called disjunction. At least one chiasma forms between each epithelium, this surface faces the basal lamina. The remaining surfaces
homologous pair, exchanging maternal and paternal sequences; up to are known as the lateral cell surfaces. In many instances, the lateral and
five have been observed. In the ovaries, primary oocytes become diplo- basal surfaces perform similar functions and the cellular domain is
tene by the fifth month in utero and each remains at this stage until the termed the basolateral domain. Cells actively transport substances, such
period before ovulation (up to 50 years). as digested nutrients from the intestinal lumen or endocrine secretions,
across their basal (or basolateral) surfaces into the subjacent connective
Diakinesis Diakinesis is the prometaphase of the first meiotic divi- tissue matrix and the blood capillaries within it. Dissolved non-polar
sion. The chromosomes, still as bivalents, become even shorter and gases (oxygen and carbon dioxide) diffuse freely between the cell and
thicker. They gradually attach to the spindle and become aligned at a the blood stream across the basolateral surface. Apical and basolateral
metaphase plate. In eggs, the spindle forms without centrosomes. surfaces are separated by a tight intercellular seal, the tight junction
Microtubules first nucleate and are stabilized near the chromosomes; (occluding junction, zonula adherens), which prevents the passage of
the action of various motor molecules eventually sorts them into a even small ions through the space between adjacent cells and thus
bipolar spindle. Perhaps surprisingly, this spindle is as efficient a maintains the difference between environments on either side of the
machine for chromosome segregation as the spindle of mitotic cells epithelium.
with centrosomes at the poles.
Metaphase I Cell surface apical differentiations
Metaphase I resembles mitotic metaphase, except that the bodies attach-
ing to the spindle microtubules are bivalents, not single chromosomes. The surfaces of many different types of cell are specialized to form
These become arranged so that the homologous pairs occupy the structures that project from the surface. These projections may permit


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
22
1
NOITCES
movement of the cell itself (flagella), or of fluids across the apical cell its distal region, called the transition zone. The continued elongation
surface (cilia), or increase the surface area available for absorption of the cilium requires the import and intraciliary transport of tubulin
(microvilli). Infoldings of the basolateral plasma membrane also dimers to the distal tip by bidirectional motor-driven proteins of the
increase the area for transport across this surface of the cell. In most intraflagellar transport complex.
non-dividing epithelial cells, the centriole-derived basal body gives rise The constant length of cilia is maintained by a steady-state balance
to a non-motile primary cilium, which has an important mechanosen- between tubulin turnover and addition of new tubulin dimers at the
sory role. ciliary tip.
Several filamentous structures are associated with the 9 + 2 doublet
Cilia and flagella microtubule of the axoneme in the cilium or flagellum shaft, e.g. radial
Cilia and flagella are motile, hair-like projections of the cell surface, spokes extend inwards from the outer doublet microtubules towards
which create currents in the surrounding fluid or movements of the cell the central pair, surrounded by an inner sheath (see Fig. 1.17). The outer
to which they are attached, or both. There are two categories of cilia: doublet microtubules bear two rows of tangential dynein arms attached
single non-motile primary cilia and multiple motile cilia. Primary cilia to the complete A subfibre of the doublet (consisting of 13 protofila-
are immotile but can detect physical and biochemical signals. Motile ments), which point towards the incomplete B subfibre of the adjacent
cilia are present in large numbers on the apical epithelial domain of doublet (consisting of 10–11 protofilaments). Adjacent doublets are
the upper respiratory tract and oviducts, and beat in a wave-like motion also linked by thin nexin filaments. Tektins are scaffolding filamentous


to generate fluid movement. Cilia also occur, in modified form, at the proteins extending along the axonemal microtubules.
dendritic endings of olfactory receptor cells, vestibular hair cells (kino- In motile cilia, arrays of dynein arms with ATPase activity cause outer
cilium), and the photoreceptor rods and cones of the retina. Flagella, microtubule doublets to move past one another, resulting in a large-
with a primary function in cell locomotion, are found on single-cell scale bending motion. Microtubules do not change in length. Move-
eukaryotes and in spermatozoa, which each possess a single flagellum ments of cilia and flagella are broadly similar. In addition to the
70 µm long. axoneme, spermatozoan flagella have outer dense fibres and a fibrous
A cilium or flagellum consists of a shaft (0.25 µm diameter) consti- sheath surrounding the axoneme. Flagella move by rapid undulation,
tuting most of its length, a tapering tip and a basal body at its base, which passes from the attached to the free end. In human spermatozoa,
which lies within the surface cytoplasm of the cell (Fig. 1.17). Other there is an additional helical component to this motion. In cilia, the
than at its base, the entire structure of the cilium is covered by plasma beating is planar but asymmetric. In the effective stroke, the cilium
membrane. The core of the cilium is the axoneme, a cylinder of nine remains stiff except at the base, where it bends to produce an oar-like
microtubule doublets that surrounds a central pair of single microtu- stroke. The recovery stroke follows, during which the bend passes from
bules (see Fig. 1.17). Ciliogenesis of primary cilia and motile cilia base to tip, returning the cilium to its initial position for the next cycle.
involves distinct steps. A centriole-derived basal body migrates to the The activity of groups of cilia is usually coordinated so that the bending
apical cell domain and axonemal microtubule doublets emerge from of one is rapidly followed by the bending of the next and so on,
Dynein ‘arms’
Inner sheath
Central


microtubules
Rootlet
Fig . 1 .17 A, The structure of a cilium shown in longitudinal (left) and transverse (right) section . A and B are subfibres of the peripheral microtubule
doublets (see text); the basal body is structurally similar to a centriole, but with microtubule triplets . B, The apical region of respiratory epithelial cells,
showing the proximal parts of three cilia sectioned longitudinally, anchored into the cytoplasm by basal bodies (BB) . Other cilia project out of the plane
of section and are cut transversely, showing the ‘9 + 2’ arrangement of microtubules . (B, With permission from Young B, Heath JW . Wheater’s Functional
Histology . 4th ed . Edinburgh: Elsevier, Churchill Livingstone; 2000 .)
A B
B Nexin-linking
protein
A
Tubulin subunits
Radial spoke
Microtubule
doublets
Plasma membrane
Basal body
Microtubule
triplets
BB
BB


Basic structure and function of cells
22.e1
1
RETPaHC
As indicated on page 15, the IFT-B protein complex participates in
intraciliary/intraflagellar anterograde transport of cargoes, a step essen-
tial for the assembly and maintenance of cilia and flagella; the IFT-A
protein complex is required for retrograde transport of cargoes to the
cell body for turnover. The movement of IFT proteins along microtu-
bules is catalysed by kinesin-2 (towards the ciliary tip; anterograde
direction) and cytoplasmic dynein-2 motor proteins (towards the cell
body; retrograde direction). A cargo includes axonemal components,
ciliary/flagellar membrane proteins (including the BBSome) and ciliary
signal transduction proteins.


Cell polarity and domains
23
1
RETPaHC
resulting in long travelling waves of metachronal synchrony. These pass a very thick cell coat or glycocalyx, which reflects the presence of integral
over the tissue surface in the same direction as the effective stroke. membrane glycoproteins, including enzymes concerned with digestion
Ciliary motion is important in clearing mucus from airways, moving and absorption. Irregular microvilli, filopodia, are also found on the
eggs along oviducts, and circulating cerebrospinal fluid in brain ventri- surfaces of many types of cell, particularly free macrophages and fibro-
cles. In the node of the developing embryo, cilium-driven flow is essen- blasts, where they may be associated with phagocytosis and cell motil-
tial for determining left–right visceral asymmetry (developing ity. For further reading on the cytoskeleton of microvilli, see Brown and
patterning). Cilia also have a sensory function, determined by the pres- McKnight (2010).
ence of receptor and channel proteins on the ciliary membrane. Primary Long and branching microvilli are called stereocilia, an early misno-
cilia in the collecting ducts of the uriniferous tubule sense the flow of mer, as they are not motile and lack microtubules. An appropriate name
urine and also modulate duct morphogenesis. Cilia are essential for is stereovilli. They are found on cochlear and vestibular receptor cells,
signalling through the hedgehog pathway, a mechanism involved in where they act as sensory transducers, and also in the absorptive epi-
organizing the body plan, organogenesis and tumorigenesis in verte- thelium of the epididymis.
brates. For additional reading on hedgehog signalling and primary cilia,
see Briscoe and Thérond (2013). Intercellular junctions
There is a group of genetic diseases in which cilia beat either inef-
fectively or not at all, e.g. Kartagener’s immotile cilia syndrome. Affected The basolateral region of the plasma membrane of epithelial cells estab-
cilia exhibit deficient function or a lack of dynein arms. Males are typi- lishes junctions with adjacent cells and with structural components of


cally sterile because of the loss of spermatozoan motility, and half have the extracellular matrix. Intercellular junctions are resilient and dynamic,
an alimentary tract that is a mirror image of the usual pattern (situs and prevent epithelial tissues from dissociating into their component
inversus), i.e. it rotates in the opposite direction during early develop- cells. In adults, the epidermis withstands imposed deformations because
ment. Defects in ciliary motility disrupt airway mucus clearance, leading of the interplay of two components of intercellular junctions, the junc-
to chronic sinusitis and bronchiectasis. Defects in sensory cilia deter- tional cytoskeleton and cell adhesion molecules (Fig. 1.19). The estab-
mine polycystic kidney disease, anosmia and retinal degeneration. lishment and maintenance of cell polarity in an epithelial layer depends
on two circumferential apical belts, the tight junctions and the zonulae
Microvilli
adherentes, running in parallel to each other and associated with
Microvilli are finger-like cell surface extensions usually 0.1 µm in diam-
F-actin. These two belts control epithelial permeability and determine
eter and up to 2 µm long (Fig. 1.18).
epithelial cell polarity. The apical cell domain resides above the belts;
Microvilli are covered by plasma membrane and supported inter-
the basolateral cell domain resides below the belts. Desmosomes
nally by closely packed bundles of actin filaments linked by cross-
(maculae adherentes) are a third class of spot-like intercellular adhe-
bridges of the actin-bundling proteins, fascin and fimbrin. Other
sion. In contrast to tight junctions and the zonulae adherentes, desmo-
bridges composed of myosin I and calmodulin connect the filament
somes do not form belts and link instead to intermediate filaments. The
bundles to the plasma membrane. At the tip of each microvillus, the
hemidesmosome, anchoring epithelial cells to the basal lamina, also
free ends of microfilaments are inserted into a dense mass that includes
links to intermediate filaments. Gap junctions are unique: they provide
the protein, villin. The actin filament bundles of microvilli are embed-


direct connection between adjacent cells and are not linked to the
ded in the apical cytoplasm amongst a meshwork of transversely
cytoskeleton. Molecular aspects of cell adhesion molecules will be con-
running actin filaments stabilized by spectrin to form the terminal web,
sidered first and then integrated with the junctional cytoskeleton to
which is underlain by keratin intermediate filaments. The web is
define specific structural and molecular aspects of different intercellular
anchored laterally to the tight junctions and zonula adherens of the
junctions.
apical epithelial junctional complex. Myosin II and tropomyosin are
also found in the terminal web, which may explain its contractile Cell adhesion molecules
activity. Cell adhesion molecules are transmembrane or membrane-anchored
Microvilli greatly increase the area of cell surface (up to 40 times), glycoproteins that bridge the intercellular space from the plasma mem-
particularly at sites of active absorption. In the small intestine, they have brane to form adhesive contacts. There are a number of molecular
subgroups, which are broadly divisible on the basis of their dependence
on calcium for function. Calcium-dependent cell adhesion molecules
include cadherins and selectins. Calcium-independent cell adhesion
molecules include the immunoglobulin-like superfamily of cell adhe-
sion molecules (Ig-CAMs), including nectins, and integrins, the only
cell adhesion molecules consisting of two subunits (α and β subunits).
Calcium-dependent cell adhesion molecules:
cadherins and selectins
Cadherins are single-pass transmembrane glycoproteins, with five
heavily glycosylated calcium-binding external domains and an intra-
cellular catenin-binding cytoplasmic tail. Catenins are intracellular
proteins linking cadherins to F-actin in the belt-arranged zonula adhe-
rens. The extracellular segment of cadherins participates in Ca2+-depend-
ent homophilic trans-interactions in which a cadherin molecule on one
cell binds to an identical cadherin molecule on an adjacent cell. After
binding, cadherins cluster laterally (cis-interaction) at cell–cell junc-
tions to form a zipper-like structure that stabilizes tight adhesion
between cells.
Different cell types possess different members of the cadherin family,


e.g. N-cadherins in nervous tissue, E-cadherins in epithelia, and
P-cadherins in the placenta. Two further members of the cadherin
family are the desmogleins and the desmocollins. Cadherins are present
in macula adherens and desmosomes but not in tight junctions or
hemidesmosomes (see below). Alterations in the expression of cadher-
ins in the epidermis produce pathological conditions such as blisters
and ulcerations. See Brieher and Yap (2013) for further reading on
cadherins and their associated cytoskeleton.
As with cadherins, selectins are Ca2+-dependent. In contrast to cad-
Fig . 1 .18 Microvilli sectioned longitudinally in the striated border of an
herins, selectins do not establish homophilic trans-interactions. Instead,
intestinal absorptive cell in a human duodenal biopsy specimen . Actin
filaments fill the cores of the microvilli and insert into the apical they bind to carbohydrates and belong to the group of lectins. Each
cytoplasm . A prominent glycocalyx (formed by the extracellular domains selectin has an extracellular carbohydrate recognition domain (CRD)
of plasma membrane glycoproteins) is seen as a fuzzy coat at the tips of with binding affinity to a specific oligosaccharide attached to a protein
and between microvilli; it includes enzymes concerned with the final or lipid. The molecular configuration and binding affinity of the CRD
stages of digestion . (Courtesy of Dr Bart Wagner, Histopathology to carbohydrate moieties is Ca2+-dependent. Selectins participate in
Department, Sheffield Teaching Hospitals, UK .) the homing of leukocytes circulating in blood towards tissues by


Basic structure and function of cells
23.e1
1
RETPaHC
When arranged in a regular parallel series, as typified by the absorp-
tive surfaces of the epithelial enterocytes of the small intestine and the
proximal convoluted tubule of the nephron of the kidneys, microvilli
acquire a fuzzy appearance like the bristles of a paintbrush (the designa-
tions brush border or striated border are used at the light microscope
level).
The cytoplasmic tail recruits proteins of the catenin complex:
β-catenin is the first to be recruited and the cadherin–β-catenin complex
rapidly recruits α-catenin; α-catenin binds directly to F-actin and coor-
dinates the activity of actin nucleating proteins and actin binding part-
ners (such as vinculin and α-actinin) to provide the dynamic forces to
modulate cell–cell adhesion; p120-catenin binds to the cytoplasmic tail
of cadherin and becomes a positive regulator of cadherin function.


BaSIC STRuCTuRE aNd fuNCTION Of CEllS
24
1
NOITCES
Claudin
Occludin Afadin–nectin complex
APICAL DOMAIN
Catenin complex ZO-1, ZO-2 and ZO-3 Tight
(occluding)
junction
Tight (occluding) junction
Afadin–nectin complex
Zonula
Zonula adherens Actin adherens Tight (occluding) junction
Cadherins
Macula adherens Intermediate
filaments Gap
Plakoglobin, plakophilin Cadherins junction
and desmoplakin
Ig-CAMs
Selectins
Intermediate filaments
Talin Actin
Hemidesmosome
Vinculin
Integrin
Integrin
Macula
S S
Fibronectin Perlecan adherens
S S
Laminin
Type IV collagen
Collagens
Nidogen (entactin)
Fig . 1 .19 Intercellular junctions: the apical junctional complex and other junctional specializations, illustrating the protein components of each junction
and of the basal lamina . An anastomotic network of contacts between adjacent cell membranes forms a tight occluding junction . Basal plasma
membrane is attached to a basal lamina at a hemidesmosome . In a gap junction, numerous channels (pores within connexons) are clustered to form a
plaque-like junctional region between adjacent plasma membranes . (A and C are transmission electron micrographs; B and D are freeze-fractured
preparations .) A, An apical junctional complex . B, A tight junction . C, A hemidesmosome . D, A gap junction . (B, Courtesy of Dr Andrew Kent, King’s
College London . D, Courtesy of Professor Dieter Hülser, University of Stuttgart . A,C, From human tissue, courtesy of Dr Bart Wagner, Histopathology
Department, Sheffield Teaching Hospitals, UK . Diagram modified from Kierszenbaum AL, Tres LL . 2012 . Histology and Cell Biology: An Introduction to
Pathology . 3rd ed, Philadelphia: Elsevier, Saunders; 2011 .)
extravasation across the endothelium. For additional reading on the loops, a transmembrane segment and a cytoplasmic tail. The nectins


significance and mechanism of homing, see Girard et al (2012). and Necls consist of four and five members, respectively. These are
Three major types of selectin include L-selectin (for lymphocytes), present in the belt-like tight junctions and zonula adherens.
E-selectin (for endothelial cells) and P-selectin (for platelets). The nectin–afadin complex initiates the formation of a zonula
adherens and after cell–cell contacts are formed between adjacent
Calcium-independent cell adhesion molecules:
cells, cadherins are recruited to these contact sites. Afadin and α-catenin
Ig-Cams, nectins and integrins interact with one another and also with F-actin through adaptor
Ig-CAMs are cell-surface glycoproteins with an extracellular domain proteins.
characterized by a variable number of immunoglobulin-like loops. Integrins mediate cell–extracellular matrix and cell–cell interactions,
Most Ig-CAMs have a transmembrane domain; others are attached to and integrate extracellular signals with the cytoskeleton and cellular
the cell surface by a glycophosphatidyl inositol (GPI) anchor. As in signalling pathways. Because integrins can be activated by proteins
cadherins, Ig-CAMs establish homophilic interactions contributing to binding to their extracellular or their intracellular domains, they can
cell–cell adhesion, although in a Ca2+-independent manner. The cyto- function in a bidirectional fashion by transmitting information
plasmic tail of Ig-CAMs also interacts with cytoskeletal components outside-in (cues from the extracellular environment) and inside-out
such as F-actin, ankyrins and spectrin. Ig-CAMs can directly or indirectly (cues from the intracellular environment) of the cell. The integrin
bind growth factor receptors and control their internalization. family of proteins consists of α subunits and β subunits forming trans-
Different types are expressed in different tissues. Neural cell adhe- membrane heterodimers. The amino-acid sequence arginine–glycine–
sion molecules (N-CAMs) are found on a number of cell types but are aspartic acid, or RGD motif, on target ligands (such as fibronectin,
expressed widely by neural cells. Intercellular adhesion molecules laminin and other extracellular matrix proteins) has binding affinity


(ICAMs) are expressed on vascular endothelial cells. Cell adhesion to the extracellular binding head of integrins. For further reading on
molecule binding is predominantly homophilic, although some use a integrins and their ligands properties, see Barczyk et al (2010).
heterophilic mechanism, e.g. vascular intercellular adhesion molecule The actin-binding protein talin binds the cytoplasmic domain of
(VCAM), which can bind to integrins. integrin β subunit and activates integrins. Vinculin interacts with talin
Nectins and nectin-like molecules (Necls) are members of the and α-actinin cross-links two filaments of actin. Kindlins, named after
Ig-CAM superfamily (see Takai et al (2008) for further reading on the gene mutated in Kindler’s syndrome, a skin blistering disease, inter-
nectins and Necls). They have an extracellular domain with three Ig-like act with talin to activate integrins.
NIAMOD
LARETALOSAB
ANIMAL
LASAB
XELPMOC
LANOITCNUJ
LACIPA
A B
C D
Hemidesmosome Gap junction


Basic structure and function of cells
24.e1
1
RETPaHC
Homing, a process that also enables thymus-derived T cells (see
Ch. 4) to home in on lymph nodes, consists of two phases. In the first,
selectin phase, carbohydrate ligands on the surface of leukocytes adhere
loosely to selectins present on the surface of endothelial cells. During
the second, cooperative sequential integrin phase, strong adhesion
permits the transendothelial migration of leukocytes into the extravas-
cular space in cooperation with cell adhesion molecules of the Ig-CAM
superfamily.
Nectins can interact homophilically or heterophilically with other
nectins to mediate, primarily, adhesion. The intracellular domain of
nectins binds to the cytoplasmic adaptor protein afadin, which links to
actin, whereas Necls interact with scaffolding proteins but not to afadin.
Necls are involved in a large variety of cellular functions, including
axon–glial interaction, Schwann cell differentiation and myelination.
In humans there are about 18 α-subunit subtypes and 8 β-subunit
subtypes, which produce 24 integrin heterodimers. The subunits are
associated by non-covalent interactions and consist of an extracellular
ligand-binding head, two multidomain segments, two single-pass trans-
membrane segments and two cytoplasmic tails. Upon binding of extra-
cellular ligands, integrins undergo a conformational change (integrin
activation), which allows the recruitment of several cytoplasmic F-actin
activator proteins (such as talin, vinculin, α-actinin and kindlins) to
their short cytoplasmic domain. This results in the formation of a
protein complex that interacts with the actin cytoskeleton.
In addition, the protein complex promotes the recruitment and
activation of several protein kinases (such as focal adhesion kinase),
leading to the activation of signalling pathways essential for several
cellular activities such as cell migration, proliferation, survival and gene
expression.