Cell Exam 4

Function of the cytoskeleton

provide structure and support for cells, determine shape, position organelles. Tracks for intracellular transport and trafficking. Generate force, contraction, movement. Cell division.

main types of cytoskeleton filaments

actin/micro filaments, microtubules, intermediate filaments

characteristics of actin/micro filaments

associate with the cell cortex under the plasma membrane. Shorter, thinner, and more dynamic

characteristics of microtubules

originate from a MTOC, grow radially towards the plasma membrane. main tracks for movement, fairly long and large. hollow, stiff, and rigid for mechanical support.

characteristics of intermediate filaments

not dynamic and are stable, fairly flexible ropes that are resistant to stretching. Help cells deal with mechanical stress. in both the cytoplasm and the nucleus, where they help to form the nuclear envelope

filament assembly

free floating subunits assemble into protofilaments, which interact laterally through non-covalent interactions to form filaments. lateral assembly helps stabilize the filament.

nucleation

first step in filament formation. a few filaments interact to form small groups, is the rate limiting step.

elongation

rapid growth of the filament

treadmilling

ability of actin filaments to have addition and removal on both sides, so add on one, remove on the other to move.

microtubule building blocks

made of dimeric building blocks with alpha and beta tubulin subunits. piled up to form a protofilament. Because of the alpha-beta pattern, microtubules are polarized, with a plus and minues end.

alpha tubulin

binds GTP structually

beta tubulin

binds GTP and can hydrolyze it into GDP (means there are both T and D forms)

Microtubule organizing center (MTOC)

area where nucleation of microtubules occurs. most common forms are centrosomes and basal bodies

centrosomes

two centriole barrels at 90 degrees to each other. 1 pair per cell. Located in the middle of the cell. minus end of the microtubule is always associated with the centrosome.

basal bodies

reside at the base of the cilia or flagella. identical in structure to the centrioles, help with the movement of those structures through microtubules.

actin building blocks

made of actin protein monomers, that can bind and hydrolyze ATP (T and D form). two actin monomers polymerize into a filament. bind head to tail so are polarized

intermediate filament types

nuclear localized (lamins) which give structure to the nuclear envelope and organize nuclear pores. Cytoplasmic intermediate filaments, which are present in the rest of the cell

how are intermediate filaments connected to other cytoskeletal elements?

plectin bridges

intermediate filament building blocks

dimers that associate with each other to form tetramers, which build symmetrical, nonpolar filaments with identical middle coils. Do not bind GTP or GDP. since they are nonpolar, motor proteins cannot walk on them.

critical concentration

the concentration of free subunits vs polymerized ones. the concentration of free subunits at which there is not net change in filament size (association = disassociation)

association rate

= K on * concentration of free subunits

dissociation rate

= K off (concentration doesn't matter)

how do Kon and Koff differ at different places

the ends of filaments have different Koff and Kon, and a different Cc due to their different properties. T and D forms also have different ones.

high critical concentration =

harder to grow, easier to shrink

how do ATP (actin) or GTP (microtubules) affect end behavior

soluble subunits are almost always in the T form, and polymerized subunits can be T or D. being added to the filament turns on hydrolysis to change from T to D. The D form has a higher Cc, so it is more likely to dissociate

life cycle of a subunit

free subunit in T form --> added to a filament --> T to D form --> dissociates

how to microtubules and actin differ in their growth/shrinkage

in microtubules, all growth and shrinkage happens at the + end. Actin can grow and shrink at both ends, but growth is favored at the plus end.

how do subunit dynamics differ at the minus and plus end of actin

minus end additions slow, so hydrolysis catches up. Plus end addition is fast, so hydrolysis lags behind.

dynamic instability in microtubules

the minus end of the filament is stable at the MTOC, but a catastrophic event can occur at the plus end, causing it to shrink rapidly towards the MTOC. if the T form cap at the plus end is lost, it will cause the filament to curve (because D forms are naturally curve) and rapidly dissociate.

proteins that regulate nucleation in actin

arp2/3 complexes, formin

arp 2/3 complexes

produce branched actin networks. arp 2 and 3 form a dimer complex that acts at the minus end to allow two subunits of actin to bind to a preexisting actin filament.

formin

produces linear, unbranched actin filaments. associates at the plus end to add one actin subunit at a time using a ring space.

proteins that can regulate the elongation phase

thymosin, profilin

thymosin

prevents addition in the elongation phase

profilin

encourages addition in the elongation phase

proteins that can regulate completed filaments

cofilin, capping proteins

cofilin

destabilized filaments by inducing a twist between the subunits, causing strain. binds to ADP-actin to promote turnover of older subunits

capping proteins

stabilize completed filaments by preventing ATP/GTP hydrolysis and physically preventing dissociation. Allows for growth at the minus end only. Plays a role in stabilizing muscle filaments

role of motor proteins

carry membrane bound organelles/vesicles to destinations in the cell. Allow for the sliding of filaments to drive contraction in cilia/flagella and muscles. can only move one direction along a filament.

function of motor proteins

covert ATP into mechanical energy to generate force. Hydrolysis provides energy to drive a conformation change. The pairing of the motor domain (heads) that hydrolyze ATP with a lever domain allows for amplification of movement.

main types of motor proteins

myosin, kinesin, dynein

myosin

associate with actin filaments. types that move towards both the minus and plus ends. big role in muscle contraction

kinesin

associated with microtubules, most move - --> + (towards plus end). play a big role in cell division and separation of sister chromatids.

dynein

associated with microtubules, most move + --> - (towards minus end). play a big role in the beating of cilia and flagella.

characteristics of a motor protein

velocity and processivity (how long a motor protein head stays associated and active with a filament). usually better at one than the other.

how does ATP/ADP form affect kinesin heads

ADP bound has low affinity for the filament, ATP bound has high affinity.

structure of the skeletal muscle

a muscle cell is made of the fused cytoplasms of several muscle cells. Inside are many myofibrils, which are made up of sarcomeres.

sarcomere

contractile unit of the muscle. composed of light and dark bands (actin and myosin respectively), the middle of the light band is the Z-disc, and a sarcomere is the distance between two Z-discs.

important proteins in sarcomeres

CapZ, tropomodulin, nebulin, titin, troponin, and tropomyosin

CapZ

attaches the plus side of the actin filament to the Z Disc.

tropomodulin

cap at the minus end of the actin filament

nebulin

binds actin and determines the length of the actin filament.

titin

positions the myosin thick filament. shaped like a spring and facilitates the return to normal distance after contraction.

troponin

protein complex that helps position tropomyosin on the actin filament. what calcium binds to

tropomyosin

elongated protein that wraps around the actin filament, covering the binding sites for myosin heads.

how does myosin move in muscle contraction

two independent (non-coordinated) heads that are plus end directed motors (move towards the Z disk). low processivity and high velocity.

how does the cytoplasm and ER interact with sarcomeres

the plasma membrane T-tubules wrap around the sarcomeres, along with the smooth ER (called the sarcoplasmic reticulum). this allows nerve impulses to reach the sarcomeres.

steps for muscle contraction

muscle cell receives an action potential. triggers the release of calcium from the sarcoplasmic reticulum. calcium binds to troponin, which causes the loss of the tropomyosin-troponin interaction, allowing the tropomyosin to move and uncover the myosin binding sites on actin. myosin heads bind to the actin filaments and contract.

what is the structure of the cytoskeletal features of flagella and cilia

called axonemes. Made of doublets of microtubules arranged around two central ones. attached to the doublets are dynein motor proteins with the tail attached to one and the heads 'looking' towards the next. all doublets are connected by cross linking proteins that prevents sliding.

how do microtubules cause cilia and flagella beating

since all of the doublets are connected, the walking of the dynein proteins causes the microtubules to bend instead, allowing them to beat.

why do cells migrate/crawl

during development to be in the proper location, to migrate to a wound for healing and fighting infections, to feed (single celled organisms), metastatic cancer cells migrate.

which type of filament is used in cell crawling

actin

how do cells crawl?

leading edge of the cell builds a protrusion with actin polymerization. Protrusion makes contact with and attaches to the substrate. back side of the cell release and the cell body is squeezed by myosin II to move the cell forward.

type of protrusions for cell crawling

filopodia (spikes, and lamellipodia (broad, flat extensions)

how are fliopodia built

tight bundle of parallel, unbranched actin. involved formin and profilin for building

how are lamellipodia built

branched actin network built by Arp2/3 nucleating at the leading edge. at the cytoplasmic side, actin disassembly is mediated by cofilin.

the extracellular matrix (ECM)

glue that holds cells together. Provides physical and biochemical signals that play key regulatory roles in determining the function of a cell. acts as a substratum for cell movement.

basal lamina/basement membrane

organized sections of the ECM that surrounds nerve fibers, muscles, fat cells, and underlies the surface of epithelial tissues and blood vessels. provides mechanical support, generates signals, is a barrier to the passage of macromolecules.

what are the main components of the ECM

collagen, proteoglycans, fibronectin, laminins

collagen

most abundant protein in the human body. trimers that form a rod-like triple helix form rigid fibrils that organize into fibers. contain hydroxylated prolines and lysines, which are key for rigidity and stability, resistant to pulling forces.

proteoglycans

proteins that have been highly modified by sugars. are the 'sticky' part of the ECM. can be assembled into large complexes through linkage of their cores onto scaffolds (like hyaluronic acid)

fibronectin

master organizer. elongated dimer of two polypeptides. has different domains that bind to different molecules, including cell receptors for cell adhesion.

laminins

preforms as similar job to fibronectin, but much smaller with three subunits. binds to cell surface adhesion receptors and other components of the ECM

integrin proteins

mediate cell-ECM attachments. are transmembrane proteins that interacts with the ECM on the outside of the cell and cytoskeletal components on the interior (F-actin or intermediate filaments)

integrin protein structure

made of a dimer of an alpha and beta chain. have a large domain outside of the cell and smaller intercellular domain. can be found in an active (open) form where it interacts with the ECM, or the inactive (closed) form where it does not. also play a role in cell signaling, especially inside-out signaling

inside-out signaling

done by integrin and cadherin proteins. change inside the cell triggers something outside of the cell.

focal adhesions

discrete sites that anchor cells to the substratum. large cluster of integrin proteins and others that are connected to actin filaments inside of the cell. can create and respond to mechanical forces, signaling hub. present in epithelial and ECM cells

hemidesmosomes

discrete anchoring sites formed of a large cluster of integrins attached to intermediate filaments. anchor the basal surface of epithelial cells to the basement membrane. capable of signaling, resist strong forces. Only in epithelial cells.

cadherin proteins

mediate cell-cell attachment. transmembrane proteins (f-actin or intermediate inside through catenins) that bind to others of the same type to bind cells together. requires Ca2+ for binding, as it decreases cadherin flexibility. specific cell types tend to have specific types of cadherin that bind together for tissue organization. can do signaling, including in-out.

catenin

protein that connects cadherin proteins with the cytoskeleton.

adherens junctions

discrete sites composed of cadherin clusters, connected to actin filaments

desmosomes

discrete sites of cadherin clusters (especially demogleins and desmocollins) that anchor to intermediate filaments.

occluding/tight junctions

make epithelial cells impermeable by occurring at the apical domain to stop things from entering the epithelium between cells. prevent transmembrane proteins from diffusing out. made by claudin and occulin proteins.

channel-forming/gap junctions

link cell cytoplasms to allow for the passage of small molecules between cell membranes. allows for very quick signaling for cell coordination/synchronization (think heart). made by connexin proteins.

signal relaying junctions (synapses)

allow signaling events to pass from cell to cell.

stages of the cell cycle

G1 (growth), S (DNA replication/synthesis), G2 (growth), and M (mitosis). not every cell has to go through every phase and the G phases can vary in length.

role of cyclins in the cell cycle

cyclins are special protein factors that accumulate in the cell to act as a time and signal for different stages in the cell cycle. When they reach a certain threshold they activate cyclin dependent kinases (CDKs). cyclin amounts are regulated by hundreds of pathways involving nutrients, hormones, etc.

G0 phase

cells can exit the cell cycle and go into G0, where the cell is no longer actively dividing. can be permanent or temporary depending on cell type.

S phase

replication of the entire genome exactly once.

how is DNA replicated exactly once at the S-phase

starts at origins of replication along the genome to copy shorter sections and then join them. Pre-RC complexes are recruited to the origins of replication, and during the S-phase those complexes will recruit a preinitiation complex, which includes the helicase for opening. As soon as the DNA polymerase starts to copy DNA is disassembled the pre-RC complex, meaning thar replication cannot happen again until after another complex has been recruited in G1.

mitotic spindle

structure made of microtubules used in mitosis. Originates from the centrosome, which has been duplicated. made of astral, interpolar, and kinetochore microtubules.

astral microtubules

contact the cell cortex and reach the plus end towards the plasma membrane. Dynein motor proteins walk along these to pull the centrosomes apart.

interpolar microtubules

reach towards the middle of the cell, in the equatorial region. Kinesin 5 and Kinesin 14 walk here.

Kinetochore Microtubules

attach to the chromosomes at the centromere and mediate their movement.

kinetochore

mediate attachment of microtubules to centromeres on chromatids. collar shape around the microtubules.

how to motor proteins and microtubules interact to pull apart sister chromatids?

Dynein motor proteins walk towards the minus end of astral microtubules, from where they are attached to the cell cortex. Kinesin 5 is plus end directed and walks on opposite interpolar microtubules to pull them apart. Kinesin 14 is minus end directs and is connected to one microtubule and walks on another to bring centrosomes back together if needed. Kinetochore proteins use dynamic instability to rapidly disassemble and slide the kinetochore and chromosomes towards the cell edges.

prophase

chromosomal condensation and untangling, spindle formation

prometaphase

nuclear breakdown and spindle attachment through kinetochores

metaphase

chromosomal alignment. determines if the right tubules have been attached to the right sister chromatids by pulling and looking for tension from opposite tubules.

anaphase

separation of the sister chromatids. activation of the motor proteins and dynamic instability