Cytoskeleton (Sec 2 Week 4)

Major functions of cytoskeleton (4)

1. Structural Support

2. Internal organization of cell (organelles and vesicle transport)

3. Cell division

4. Large scale movement

3 types of protein filaments and their diameter (AMI)

1. Actin filaments, 7 nm

2. Microtubules, 10 nm

3. Intermediate filaments, 25 nm

Microscopy techniques to view cytoskeleton (3

• Light microscope - basic light, limited resolution 200 nm

• Fluorescence - Detects specific fluoresced proteins

• Transmission electron - uses beams of electron, high resolution

Immunofluorescence purpose

• Determine location of protein in cell

• Uses two antibodies (First binds to cell, second binds to first and is visible when fluoresced)

Intermediate filament purpose and 2 types (Cy + N)

Involved in structural support

cytoplasmic - in animal cells subject to stress- provides mechanical strength

Nuclear - in all animal cells, in nucleus to provide regulation and support

Structure of cytoplasmic intermediate filaments (3)

a-helical central rod

2 monomers wrap together into coiled-coil, 2 of the dimers a staggered antiparallel tetramers

No polarity bcause tetramer ends are same

IF in epithelial cell

Keratin filaments anchoed at cell-cell junctions

Microtubules function

• organizing function in all eukaryotes

• Mitosis, structural support

Tubulin structure (4)

long stiff hollow inextensible tubes

Made of individual subunits (a + b tublin form a heterodimer bound to GTP)

Heterodimers are polar (b = +, and a = -)

13 parallel protofilaments make up the hollow tube

MT protofilaments

bonds are noncovalent

Bonds between weaker than bonds within

Growth of MT

occurs at both ends, faster at plus end

Microtubule Organizing Centers (MTOC)

microtubules grow from MTOCs (- end is attched to MTOC), they are in dynamic instability for remodeling

Dynamic Instability: Growth

ab-tubulin dimers added to growing plus end

GTP becomes hydrolyzed into GDP, but addition of new GTP is faster than hydrolysis so GTP cap appears

Dynamic Instability: Shrinking

If ab-tubulin dimers added at slower rate than hydrolysis, then no GTP cap and the weaker GDP-tubulin dissasembles

Dynamic Instability Mechanism

a-tubulin GTP is not hydrolyzed, b-tubulin is

GTP cap is straighter, stronger binding favouring growth in comparison to GDP-tubulin dimer

MTOC functions

1. nucleating sites for MT growth (eg y-tubulin ring complex acts as attachment site for inital tubulin dimers)

In nondividing animals in interphase, most MT radiate from…

one MTOC, two in dividing.

Goals of microtubule-associated proteins (4) NPDS

1. nucleate new growth

2. microtubule polymerization

3. dissasembly

4. stabilize microtubules

Motor Proteins and intracellular transport

Kinesins move towards plus end (makes sense its the part that grows out) - uses ATP hydrolysis for movement through head, uses tail to transport

Dyneins - move towards minus end (backward transport)

Actin filaments properties

• made of actin monomers

• flexible, inextensible

Actin F functions (4) SCMC

• Stiff structures

• Contractile activity

• Motility

• cytokinesis (Contractile ring)

Actin filament structure

Helical, composed of globular protein monomers, polar ends

Actin monomers (3)

1. Free monomers are bound to ATP

2. Hydrolysis ATP to ADP, reducing strength of binding

Actin polymerization in vitro (growth trend)

Reaches horizontal equilibrium as the largest actin filament elongates concentration of monomers decreases, decreasing growth rates until eq

treadmilling

ACtin filament minus end does not grow fast enough to outpace dissasembly caused by hydrolysis, but positive end can still grow - causes treadmill motion with actin monomers moving through filament and eventually getting replaced

Myosins and myosin 1 vs myosin 2

Actin motor proteins that move towards plus end using ATP hydrolysis for movement

myosin 1: Tail binds cargo

myosin 2: Dimer with a coiled tail acts as a contractile force