Cell Structure & Function Exam IV

cytoskeleton

gives structure and form to cells, primary way that cells generate force

What are some of the processes that the cytoskeleton is used to drive? (5)

cell division, trafficking of organelles, cell contraction in muscle cells, cell movement & migration, beating of cilia

three types of cytoskeletal filaments

actin filaments, microtubules, intermediate filaments

actin filaments

• associated with cell cortex (PM)

• shorter, smaller, and more flexible than microtubules

• drive contraction movements (muscle and cell division)

microtubules

• in cytoplasm, often emanating out of a centrosome

• long, hollow cylinders

• rigid structure

• can serve as highways for organelle and chromosome movement

What does MTOC stand for? Give an example

microtubule organizing center, ex: centrosome

intermediate filaments

• in cytoplasm and nucleus

• act as fairly flexible ropes that are resistant to stretching

• function to help cells deal w/ mechanical stress and not be torn apart

Examples and short description of how cytoskeletal filaments form stable structures and direct polarity (2)

stereocilia: actin filaments in the ear that are formed once and hopefully last a lifetime

epithelial cells: has stable F-actin and microvili and microtubules that direct apical basal polarity

describe how smaller subunits assemble into cytoskeletal filaments

often assemble into a helical structure through non-covalent interactions

Why do cytoskeletal filaments assemble through non-covalent interactions?

the subunits are more weakly held together so they can break down and reassemble quickly

describe how protofilaments are made and what they do

subunits assemble into protofilaments that then can interact laterally to form filaments

How does the lateral interaction of protofilaments help filaments?

stabilizes interior of filaments while allowing dynamic change at ends

nucleation

very short polymers are unstable due to absence of stabilizing interactions, often the rate limiting step in filament formation

microtubule formation and organization

• made of heterodimers of alpha- and beta-tubulin proteins that can bind and hydrolyze GTP

• heterodimers assemble head-to-tail in protofilaments

• 13 protofilaments wrap into hollow tubes that provide rigidity

What does the assembly of heterodimers generate for microtubules?

microtubule polarity

f-actin formation and organization

• actin filaments made of actin protein monomers that can bind and hydrolyze ATP

• bends into a helical structure

• actin monomers bind in head-to-tail manner

• single and double stranded filament

What does the structure of f-actin allow for?

greater flexibility than microtubules, though can be crosslinked into stronger structures

What does the assembly of actin monomers generate for f-actin?

polarity with plus and minus ends

What may be different between the two ends of a cytoskeletal filament?

growth rates

What does it means when the subunit concentration matches a filament end’s critical concentration?

rate of subunit addition to an end is perfectly balanced with subunit subtraction

What will happen when the subunit concentration in a cell goes above the critical concentration?

filament will grow

What will happen when the subunit concentration in a cell goes below the critical concentration?

filament will disassemble

k_on

rate that subunits are loaded onto a filament

k_off

rate that subunits fall off a filament

k_off/k_on when subunit concentration equals critical concentration

=1

What controls critical concentration/filament formation?

ATP/GTP hydrolysis

What hydrolyzes ATP/GTP slowly and quickly?

individual monomers hydrolyze slowly, subunits in filaments hydrolyze quickly

What becomes more likely the longer a subunit is in a filament?

the higher likelihood it is in ADP/GDP state

What does the filament act as for the subunits?

a GAP because it inactivates them

What happens to k_off in GDP bound subunits?

it increases

describe the effects of ATP/GTP hydrolysis

• energy of hydrolysis causes conformational change that decreases filament affinity

• ADP/GDP is trapped in the filament and the energy will be released when subunits dissociate

describe how the critical concentrations of ATP/GTP vs ADP/GDP affects filament formation and what it means

critical concentration of ATP/GTP bound subunits promotes addition and the critical concentration of ADP/GDP bound subunits promotes disassembly, there is a race between hydrolysis and new subunit addition at a given end

When does treadmilling occur and what is it?

occurs when one end of the filament is largely ADP/GDP bound and the other is ATP/GTP bound, minus end disassembles and plus end assembles

What serves a “cap” on a filament?

terminal, plus end GTP in microtubules

What is the cap of a microtubule?

bunch of GTP-bound heterodimers that permit continued assembly, constrains microtubule curvature

characteristic of GDP bound subunits

have a mild curve

What happens when a microtubule cap is lost? What does this explain?

“catastrophe” - rapid depolymerization that explains the rapid switching behaviors in microtubules

Why does curving also favor disassembly?

disrupts stabilizing interactions with other protofilaments

What do almost all eukaryotic cells have and what do only some have?

almost all have microtubules and actin filaments, some have intermediate filaments

What kind of cells are intermediate filaments expressed in?

cells that deal with mechanical stress

two groups of intermediate filaments proteins

nuclear lamins, cytoplasmic keratins

nuclear localized lamins

present in all cells, give structure to nuclear envelope, organize nuclear pores

cytoplasmic intermediate filaments

formed of elongated subunits with coiled-coil domain, first bind as dimer in parallel fashion

explain how cytoplasmic intermediate filaments form and how they’re different from other cytoskeletal elements

they have no polarity, don’t bind NTPs, dimer binds another dimer in an antiparallel fashion to form tetramer

tetramer

protofilament, 8 tetramers bind to form cytoplasmic intermediate filaments

How are intermediate filaments held in place?

anchored to cell-cell contacts or the ECM

describe how cell-cell contact of intermediate filaments is directed

desmosomes direct cell-cell contact with intermediate filaments attached

describe how cell-ECM contact of intermediate filaments is directed

hemidesmosomes direct cell-ECM contact with intermediate filaments attached

Why would plants and fungi target the cytoskeleton? Give an example

halt force generating events (stop eating), ex: death cap mushrooms

describe how drugs and toxins regulate filament formation

they can bind to monomers or filaments, may activate or inhibit filament formation

describe possible effects of drugs and toxins binding monomers

may sequester monomer or promote binding to a filament

describe possible effects of drugs and toxins binding filaments

may promote or inhibit filament polymerization

How can drugs and toxins be used as scientific reagents?

to visualize or alter cytoskeletal elements, to change cytoskeletal function

Why would physicians want to change cytoskeletal function?

to target cell movement, growth, and structure to treat diseases like cancer

taxol

drug often used to treat cancer by altering cytoskeletal function

two modes of operation for regulating and positioning the cytoskeleton

regulation of filament formation/polymerization, regulation of filament after it has formed: stability and organization

regulation of filament formation/polymerization (microtubules)

occurs at the centrosome, gamma-TuRC (two accessory protein and gamma-tubulin)  provide template for filament formation

How does the gamma-TuRC complex promote formation?

provides the nucleating sites

describe how gamma-tubulin and microtubule formation is mostly restricted to the centrosome

gamma-TuRCs confined to centrosome matrix, centrosome matrix organized by centrioles, which are made of heavily modified short microtubules

two main regulators of actin nucleation

Arp2/3 complex and formin proteins

Arp2/3 complex

functions in actin nucleation by producing branched actin networks, operates through a mechanism similar to gamma-TuRC

formin proteins

functions in actin nucleation by producing linear, unbranched actin filaments, functions at plus end, formin dimer binds two actin subunits at once, processive function directs formation of linear, unbranched filament

ARP

actin related protein, act at minus end as nucleators

filament polymerization can be affected by proteins that …

bind free subunits

describe how binding free subunits can affect filament polymerization

• proteins may bind and sequester monomers, blocking their addition to a filament and decreasing the effective concentration of monomers

• proteins bind monomers and expose only the plus end binding side of actin monomer or promote ATP binding, promoting plus end assembly

severing filaments can enhance …

polymerization or disassembly

describe severing filaments

long filament can be severed into many shorter filaments

• if critical concentration favors disassembly, you get much more rapid disassembly

• if critical concentration favors assembly, you get additional templates for rapid multi-filament assembly

What are katamins and gelsolins and what do they do?

proteins, katamins cut microtubulues and require ATP, gelsolins cut F-actin and don’t require ATP

MAP

microtubule associated protein

What do MAPs do? Give two examples of MAPs

bind to microubles and stabilize them by providing additional longitudinal interactions, projecting domains can space microtubules apart, ex: MAP2 and tau

cofilin

destabilizes bound actin filaments by inducing additional twist to filament by partially inserting between actin subunits, likes to bind ADP-actin which prompts turnover of older filaments, binds 1:1 with actin subunits

describe how the twist that cofilin causes destabilizes actin filaments

twist loosens subunit interactions, terminal subunit dissociates easier, strained filament more likely to sever

two mechanisms for capping proteins stabilizing filaments

prevent ATP/GTP hydrolysis, physically prevent dissociation

Where are actin filaments capped and when?

on plus end (dynamic) after a growth period

kinesin 13

proteins that pull on microtubule protofilaments and act as microtubule depolymerases (catastrophe)

What two types of proteins can determine growth vs catastrophe of filaments?

MAPs and kinesin 13

describe the process of formation of a nucleated actin filament

activating factor is added to an inactive ARP complex, then actin monomers add from the minus to the plus end

thymosin

binds actin subunits and prevents assembly

profilin

binds actin subunits and speeds up elongation

katanin

severs microtubules

gelsolin

severs actin filaments and binds to plus end

Does the addition of cofilin to an actin filament shorten or elongate the filament?

shorten

describe the growth of uncapped filaments

growth at plus and minus ends

describe the growth of capped filaments

growth at minus ends only

describe how MAPs and kinesin 13 affect the stability of microtubules

MAP: stabilize, frequency of catastrophes suppressed and/or growth rate enhanced, results in longer, less dynamic microtubules

kinesin 13: destabilize, frequency of catastrophes increased, results in shorter, more dynamic microtubules

two ways forces are generated within a cell

filament polymerization and motor protein function

two functions of motor proteins

carry membrane bound organelles to destinations in cell, cause sliding of filaments past each other to drive contraction

What do motor proteins utilize in order to move in one direction?

filament polarity

What kind of motor would a motor protein need if an endocytic vesicle wants to go to the ER?

minus end directed motor so it will move it towards the centrosome/nucleus

How do motor proteins do physical work?

motors bind to and hydrolyze ATP

What does hydrolysis do for motor proteins?

provides energy to drive conformational changes in the protein

How do proteins motor?

motor proteins often pair a motor domain that hydrolyzes ATP, which induces a conformational change with a lever domain

role of levers in motor proteins

they amplify small conformational changes to produce large displacement

What is the lever movement couples with for motor proteins?

motor couples movement of lever with periods of filament binding and unbinding

two key characteristics of motor proteins

velocity and processivity

velocity of a motor protein

rate of physical displacement

processivity of a motor protein

how long the protein stays associate and active with a filament