Chapter 15 Cytoskeletal Systems

Cytoskeletal Systems The interior of a cell is highly structured The cytoskeleton is a network of interconnected filaments and tubules extending through the cytosol It plays roles in cell movement and division It is dynamic and changeable 2

Major Structural Elements of the Cytoskeleton The major structural elements of the cytoskeleton are Microtubules Microfilaments Intermediate filaments

Table 15-1 - Microtubules

Table 15-1 - Microfilaments

Table 15-1 – Intermediate Filaments

Eukaryotes Have Three Basic Types of Cytoskeletal Elements Indirect immunostaining has been used to characterize cytoskeletal elements Microtubules are composed of tubulin subunits and are about 25 nm in diameter Micofilaments, 7 nm wide, are composed of actin subunits Intermediate filaments, 8–12 nm, are variable in composition 7

Bacteria Have Cytoskeletal Systems That Are Structurally Similar to Those in Eukaryotes Recent discoveries show that Bacteria and Archaea have polymer systems that function similarly to eukaryotic cytoskeletal elements The actin-like MreB protein is involved in DNA segregation The tubulin-like FtsZ protein is involved in regulating division Crescentin is a regulator of cell shape 8

Figure 15-1a-d

Figure 15-1e

The Cytoskeleton Is Dynamically Assembled and Disassembled Microfilaments are essential components of muscle fibrils and microtubules are structural elements of cilia and flagella These structures are large enough to view by a variety of microscopic techniques Also, certain drugs can be used to perturb cytoskeletal function

Dynamic cytoskeleton Research has shown that the cytoskeleton is dynamically assembled and disassembled It includes some remarkably elaborate structures

Table 15-2 – first two rows . 13

Table 15-2 – remaining two rows . 14

Table 15-3

Microtubules Microtubules are the largest of the cytoskeletal components of a cell There are two types of microtubules They are involved in a variety of functions in the cell

Two Types of Microtubules Are Responsible for Many Functions in the Cell Cytoplasmic microtubules pervade the cytosol and are responsible for a variety of functions Maintaining axons Formation of mitotic and meiotic spindles Maintaining or altering cell shape Placement and movement of vesicles

Two types of microtubules (MTs) Axonemal microtubules include the organized and stable microtubules found in structures such as Cilia Flagella Basal bodies to which cilia and flagella attach The axoneme, the central shaft of a cilium or flagellum, is a highly ordered bundle of MTs

Tubulin Heterodimers Are the Protein Building Blocks of Microtubules MTs are straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments The basic subunit of a protofilament is a heterodimer of tubulin, one a-tubulin and one b-tubulin These bind noncovalently to form an ab-heterodimer, which does not normally dissociate

Figure 15-2

Figure 15-2A

Figure 15-2B

Figure 15-2C

Subunit structure a and b subunits have very similar 3-D structure, but only 40% amino acid identity Each has an N-terminal GTP binding domain, a central domain to which colchicine can bind, and a C-terminal domain that interacts with MAPs (microtubule-associated proteins) All the dimers in the MT are oriented the same way

MT polarity and isoforms Because of dimer orientation, protofilaments have an inherent polarity The two ends differ both chemically and structurally Most organisms have several closely related genes for slight variants of a- and b-tubulin, referred to as isoforms

Microtubules Can Form as Singlets, Doublets, or Triplets Cytoplasmic MTs are simple tubes, or singlet MTs, with 13 protofilaments Some axonemal MTs form doublet or triplet MTs Doublets and triplets contain one 13-protofilament tubule (the A tubule) and one or two additional incomplete rings (B and C tubules) of 10 or 11 protofilaments

Microtubules Form by the Addition of Tubulin Dimers at Their Ends MTs form by the reversible polymerization of tubulin dimers in the presence of GTP and Mg2+ Dimers aggregate into oligomers, which serve as “nuclei” from which new MTs grow This process is called nucleation; the addition of more subunits at either end is called elongation

Microtubule assembly MT formation is slow at first, the lag phase, due to the slow process of nucleation The elongation phase is much faster When the mass of MTs reaches a point where the amount of free tubulin is diminished, the assembly is balanced by disassembly; the plateau phase

Figure 15-3

Critical concentration Microtubule assembly in vitro depends on concentration of tubulin dimers The tubulin concentration at which MT assembly is exactly balanced by disassembly is called the critical concentration MTs grow when the tubulin concentration exceeds the critical concentration and vice versa

Addition of Tubulin Dimers Occurs More Quickly at the Plus Ends of Microtubules The two ends of an MT differ chemically, and one can grow or shrink much faster than the other This can be visualized by mixing basal bodies (structures found at the base of cilia) with tubulin heterodimers The rapidly growing MT end is the plus end and the other is the minus end

Figure 15-4

Microtubule treadmilling The plus and minus ends of microtubules have different critical concentrations If the [tubulin subunits] is above the critical concentration for the plus end but below that of the minus end, treadmilling will occur Treadmilling: addition of subunits at the plus end, and removal from the minus end

Figure 15-5

Drugs Can Affect the Assembly of Microtubules Colchicine binds to tubulin monomers, inhibiting their assembly into MTs and promoting MT disassembly Vinblastin, vincristine are related compounds Nocodazole inhibits MT assembly, and its effects are more easily reversed than those of colchicine

Antimitotic drugs These drugs are called antimitotic drugs because they interfere with spindle assembly and thus inhibit cell division They are useful for cancer treatment (vinblastine, vincristine) because cancer cells are rapidly dividing and susceptible to drugs that inhibit mitosis

Taxol Taxol binds tightly to microtubules and stabilizes them, causing a depletion of free tubulin subunits It causes dividing cells to arrest during mitosis It is also used in cancer treatment, especially for breast cancer

GTP Hydrolysis Contributes to the Dynamic Instability of Microtubules Each tubulin heterodimer binds two GTP molecules, a-tubulin binds one and b-tubulin binds a second The GTP bound to the b-subunit is hydrolyzed to GDP after the heterodimer is added to the MT GTP is needed to promote heterodimer interactions and addition to MTs, but its hydrolysis is not required for MT assembly

Dynamic instability Dynamic instability model: one population of MTs grows by polymerization at the plus ends whereas another population shrinks by depolymerization Growing MTs have GTP at the plus ends, and shrinking MTs have GDP The GTP cap at the plus end prevents subunit removal

Figure 15-6A

GTP-tubulin and dynamic instability If [GTP-tubulin] is high, it is added to an MT, quickly creating a large GTP-tubulin cap If the concentration falls, the rate of tubulin addition decreases At a sufficiently low [GTP-tubulin], the rate of GTP hydrolysis exceeds the rate of subunit addition and the cap shrinks

Catastrophe and rescue If the GTP cap disappears altogether, the MT becomes unstable and loss of GDP-bound subunits is favored Individual MTs can go through periods of growth and shrinkage; a switch from growth to shrinkage is called microtubule catastrophe A sudden switch back to growth phase is called microtubule rescue

Figure 15-6B

Figure 15-7

Microtubules Originate from Microtubule- Organizing Centers Within the Cell MTs originate from a microtubule-organizing center (MTOC) Many cells have an MTOC called a centrosome near the nucleus In animal cells the centrosome is associated with two centrioles, surrounded by pericentriolar material

Centriole structure Centriole walls are formed by 9 pairs of triplet microtubules They are oriented at right angles to each other They are involved in basal body formation for cilia and flagella Cells without centrioles have poorly organized mitotic spindles

Figure 15-8A,B

Figure 15-8C

g-tubulin Centrosomes have large ring-shaped protein complexes in them; these contain g-tubulin (along with gamma tubulin ring proteins: GRiPs) g-tubulin ring complexes (g-TuRCs) nucleate the assembly of new MTs away from the centrosome Loss of g-TuRCs prevents a cell from nucleating MTs

Figure 15-9

Figure 15-9A

Figure 15-9B

MTOCs Organize and Polarize the Micotubules Within Cells MTOCs nucleate and anchor MTs MTs grow outward from the MTOC with a fixed polarity—the minus ends are anchored in the MTOC Because of this, dynamic growth and shrinkage of MTs occurs at the plus ends, near the cell periphery

Figure 15-10A-C

Figure 15-10D

Microtubule Stability Is Tightly Regulated in Cells by a Variety of Microtubule-Binding Proteins Cells regulate MTs with great precision Some MT-binding proteins use ATP to drive vesicle or organelle transport or to generate sliding forces between MTs Others regulate MT structure

Microtubule-Stabilizing/Bundling Proteins MAPs, microtubule-associated proteins, bind at regular intervals along a microtubule wall, allowing for interaction with other cellular structures and filaments A MAP called Tau causes MTs to form tight bundles in axons MAP2 promotes the formation of looser bundles in dendrites

MAPs that promote bundling MAPs such as Tau and MAP2 have two regions One region binds to the MT wall and another part of the protein extends at right angles to the MT to allow for interaction with other proteins The length of the extended “arm” controls the spacing of MTs in the bundle

Figure 15-11A

+–TIP Proteins MTs can be stabilized by proteins that “capture” and protect the growing plus ends These are +–TIP proteins (+-end tubulin interacting proteins) These decrease the likelihood that MTs will undergo catastrophic subunit loss

Figure 15-11B

Microtubule-Destabilizing/Severing Proteins Some proteins promote depolarization of MTs Stathmin/Op18 binds to tubulin heterodimers and prevents their polymerization Catastrophins act at the ends of MTs and promote the peeling of subunits from the ends Proteins such as katanins sever MTs

Figure 15-11C

Microfilaments Microfilaments are the smallest of the cytoskeletal filaments They are best known for their role in muscle contraction They play a role in cell migration, amoeboid movement, and cytoplasmic streaming 64

Additional roles of microfilaments Development and maintenance of cell shape (via microfilaments just beneath the plasma membrane at the cell cortex) Structural core of microvilli

Actin Is the Protein Building Block of Microfilaments Actin is a very abundant protein in all eukaryotic cells Once synthesized, it folds into a globular-shaped molecule that can bind ATP or ADP (G-actin; globular actin) G-actin molecules polymerize to form microfilaments, F-actin

Figure 15-12

Figure 15-12A

Figure 15-12B

Figure 15-12C

Different Types of Actin Are Found in Cells Actin is highly conserved, but there are some variants Actins can be broadly divided into muscle-specific actins (a-actins) and nonmuscle actins (b- and g-actins) b- and g-actin localize to different regions of a cell

G-Actin Monomers Polymerize into F-Actin Microfilaments G-actin monomers can polymerize reversibly into filaments with a lag phase, and elongation phase, similar to tubulin assembly F-actin filaments are composed of two linear strands of polymerized G-actin, wound into a helix All the actin monomers in the filament have the same orientation

Demonstration of microfilament polarity Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs) S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern The plus end of an MF is called the barbed end and the minus end is called the pointed end, because of this pattern

Figure 15-13A,B

Figure 15-13C

Polarity of microfilaments The polarity of MFs is reflected in more rapid addition or loss of G-actin at the plus end than the minus end After the G-actin monomers assemble onto a microfilament, the ATP bound to them is slowly hydrolysed So, the growing MF ends have ATP-actin, whereas most of the MF is composed of ADP-actin

Specific Drugs Affect Polymerization of Microfilaments Cytochalasins are fungal metabolites that prevent the addition of new monomers to existing MFs Latrunculin A is a toxin that sequesters actin monomers and prevents their addition to MFs Phalloidin stabilizes MFs and prevents their depolymerization

Cells Can Dynamically Assemble Actin into a Variety of Structures Cells can regulate where and how MFs are assembled Cells that crawl have lamellipodia and filopodia at their leading edge, allowing them to move along a surface Cells that adhere tightly to the underlying substratum have organized bundles called stress fibers

Figure 15-14A

Rapidly moving cells Rapidly moving cells don’t have such striking actin bundles The cell cortex, just beneath the plasma membrane, has actin crosslinked into a gel or loose lattice

Filopodia and lamellipodia In filopodia, at the leading edge, microfilaments form highly oriented, polarized cables with the plus ends toward the tip of the protrusion The actin in lamellipodia is less well organized than in filopodia

Figure 15-14B

Figure 15-15

Actin-Binding Proteins Regulate the Polymerization, Length, and Organization of Actin Cells can precisely control where actin assembles and the structure of the resulting network They use a variety of actin-binding proteins to do so Control occurs at the nucleation, elongation, and severing of MFs, and the association of MFs into networks 84

Figure 15-16

Proteins That Regulate Polymerization If the concentration of ATP-bound G-actin is high, microfilaments will assemble until the G-actin is limiting In the cell, a large amount of free G-actin is not available because it is bound by thymosin 4 Profilin competes with thymosin 4 for G-actin binding 86

Proteins that regulate polymerization ADF/cofilin is known to bind ADP-G-actin and F-actin and is thought to increase turnover of ADP-actin at the minus end of MFs ADF/cofilin also severs filaments, creating new plus ends in the process 87

Proteins That Cap Actin Filaments Whether MFs can grow depends on whether their filament ends are capped Capping proteins bind the ends of a filament to prevent further loss or addition of subunits CapZ binds to plus ends to prevent addition of subunits there; tropomodulins bind to minus ends, preventing loss of subunits there 88

Proteins That Crosslink Actin Filaments Often, actin networks form as loose networks of crosslinked filaments One of the proteins important in the formation of these networks is filamin Filamin act as splices, joining two MFs together where they intersect 89

Proteins That Sever Actin Filaments MFs are broken up by proteins that sever and/or cap them Gelsolin breaks actin MFs and caps the newly exposed plus ends, preventing further polymerization 90

Proteins That Bundle Actin Filaments Some actin-containing structures can be highly ordered Actin may be bundled into tightly organized arrays, called focal contacts or focal adhesions -actinin is a protein that is prominent in such structures Fascin in filopodia keeps the actin tightly bundled 91

Microvilli Actin bundles in microvilli are the best-studied examples of ordered actin structures Microvilli are prominent features of intestinal mucosal cells; they increase the surface area of the cells The core of a microvillus consists of a tight bundle of microfilaments with the ends pointed toward the tip 92

Crosslinks The MFs are connected to the plasma membrane by crosslinks made of myosin I and calmodulin The MFs in the bundle are tightly bound together by crosslinking proteins fimbrin and villin 93

Figure 15-17

Figure 15-17A

Figure 15-17B

The terminal web At the base of the microvillus, the MF bundle extends into a network of filaments called the terminal web The filaments of the terminal web are composed mainly of myosin and spectrin, which connect the MFs to each other, to proteins in the membrane, and perhaps also to intermediate filaments

Figure 15-18

Proteins That Link Actin to Membranes MFs are connected to the plasma membrane and exert force on it during cell movement or cytokinesis This (indirect) connection to the membrane requires one or more linking proteins One group of such proteins is the band 4.1, ezrin, radixin, and moesin family; another is spectrin and ankyrin

Figure 15-19

Figure 15-19A

Figure 15-19B

Proteins That Promote Actin Branching and Growth Besides loose networks and bundles, actin can form a dendritic (treelike) network A complex of actin-related proteins, the Arp2/3 complex, nucleates new branches on the sides of filaments Arp2/3 branching is activated by a family of proteins that includes WASP (Wiskott-Aldrich syndrome protein) and WAVE/Scar

Figure 15-20A

Figure 15-20B

Long actin filaments For some cell functions long actin filaments are needed In this case, actin polymerization is regulated independently of the Arp2/3 complex, through proteins called formins Formins move along the end of the growing filament as they promote polymerization

Figure 15-20C

Cell Signaling Regulates Where and When Actin-Based Structures Assemble Both plasma membrane lipids and several small G proteins related to Ras regulate the formation, stability, and breakdown of MFs

Inositol Phospholipids Phosphatidylinositol-4,5-bisphosphate (PIP2) can bind to profilin, CapZ, and proteins such a ezrin PIP2 recruits these proteins to the membrane and regulates their interactions with actin CapZ binds tightly to PIP2 resulting in its removal from the end of a MF, promoting disassembly

Rho Family GTPases The cytoskeleton of cells exposed to certain growth factors can undergo a dramatic change Many signals that result in these changes act via a family of monomeric G proteins called Rho GTPases Three key family members are Rho, Rac, and Cdc42

Rho family GTPases, effect on cytoskeleton Activation of the Rho pathway results in formation of stress fibers Rac activation results in extension of lamellipodia Cdc42 activation results in the formation of filopodia

Figure 15-21A-D

Figure 15-21E

Regulation of Rho GTPases Rho GTPases are stimulated by guanine-nucleotide exchange factors (GEFs) through the exchange of bound GDP for GTP GTPase activating proteins (GAPs) inactivate Rho GTPases by causing them to hydrolyze their bound GTPs to GDP Guanine-nucleotide dissociation inhibitors (GDIs) sequester inactive Rho GTPases in the cytosol

Intermediate Filaments Intermediate filaments are the most stable and least soluble cytoskeletal components and are not polarized An abundant intermediate filament (IF) is keratin, an important component of structures that grow from skin in animals IFs may support the entire cytoskeleton

Figure 15-22

Intermediate Filament Proteins Are Tissue Specific IFs differ greatly in amino acid composition from tissue to tissue They are grouped into six classes

Classes of intermediate filament proteins Class I: acidic keratins Class II: basic or neutral keratins Proteins of classes I and II make up the tonofilaments found in epithelial surfaces covering the body and lining its cavities

Classes of intermediate filament proteins (continued) Class III: includes vimentin (connective tissue), desmin (muscle cells), and glial fibrillary acidic (GFA) protein (glial cells) Class IV: These are the neurofilament (NF) proteins found in neurofilaments of nerve cells

Classes of intermediate filament proteins (continued) Class V: includes the nuclear lamins A, B, and C that form a network along the inner surface of the nuclear membrane Class VI: Neurofilaments in the nerve cells of embryos are made of nestin Animal cells can be distinguished based on the types of IF proteins they contain—intermediate filament typing

Table 15-4

Intermediate Filaments Assemble from Fibrous Subunits IF proteins are fibrous rather than globular All have a homologous central rodlike domain conserved in size, secondary structure, and to some extent, in sequence Flanking the central helical domain are N- and C-terminal domains that differ greatly among IF proteins

Intermediate filament assembly The basic structural unit consists of two IF polypeptides intertwined into a coiled-coil The two polypeptides are aligned in parallel Two such dimers align laterally to form a tetrameric protofilament Protofilaments overlap to build up a filamentous structure about 8 protofilaments thick

Figure 15-23

Figure 15-23A

Figure 15-23B

Figure 15-23C

Figure 15-23D

Intermediate Filaments Confer Mechanical Strength on Tissues Cellular architecture depends on the unique properties of the cytoskeletal elements working together MTs resist bending when a cell is compressed whereas MFs serve as contractile elements that generate tension IFs are elastic and can withstand tensile forces

The Cytoskeleton Is a Mechanically Integrated Structure IFs are important structural determinants in many cells and tissues; they are thought to have a tension-bearing role IFs are not static structures; they are dynamically transported and remodeled The nuclear lamina, on the inner surface of the nuclear envelope, disassemble at the onset of mitosis and reassemble afterward

Integration of cytoskeletal elements Plakins are linker proteins that connect intermediate filaments, microfilaments, and microtubules One plakin, called plectin, is found at sites where intermediate filaments connect to MFs and MTs

Figure 15-24