Cytoskeletal Systems - Warner Pacific Universityclasspages.warnerpacific.edu/BDupriest/BIO 330/Ch... · Intermediate Filaments •Intermediate filaments are the most stable and least

© 2012 Pearson Education, Inc.

Lectures by

Kathleen Fitzpatrick Simon Fraser University

Cytoskeletal

Systems

Chapter 15


Table 15-1 - Microtubules


Table 15-1 - Microfilaments


Table 15-1 – Intermediate Filaments


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


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


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


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


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


Figure 15-14A


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


Figure 15-19A


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



(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



(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


Figure 15-23


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

Cytoskeletal Systems - Warner Pacific Universityclasspages.warnerpacific.edu/BDupriest/BIO 330/Ch... · Intermediate Filaments •Intermediate filaments are the most stable and least

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