Overview of the cytoskeleton., Actin, 8 types of actin proteins, Intermediate filaments, Microtubules, Myosin, Kinesin and Dynein, Cell Cycle 1, CDKs, Some Checkpoint info, G1/S transition and S phase, Mitosis and completion of division

Sequestering of actin monomers

2 main enzymes are - B-thymosin and Profilin - both exclusively bind to actin monomers (cant have both, they compete) B -thymosin - Higher affinity for ATP-actin than ADP-actin monomers - inhibits both polymerization and nucleotide exchange Profilin - binds to APO/ATP/ADP-actin monomers - stimulates nucleotide exchange - allows plus end binding - released once it binds -can displace thymosin (higher affinity)

The Dynein Power stroke

As the AAA 1 (the catalytic ATPASE) hydrolyses ATP the stock and catalytic domain undergo a rotation relative to the tail - referred to as the power stroke

Chromosomes lining up

Don't make a hard line oscillate back and forth as the spindles grow and shrink trying to get them into place

Many cells stop cycling

Enter G0 often accompanied by differentiation into a specific or specialized cell type - neurons - muscle cells In some cases these cells can leave G0 and reenter the cell cycle

Tubulin Code

Post translational modifications of tubulin within microtubules contribute to effector selection these modifications are often done to the C-terminal tails of the tubulin Different combinations lead to different functions - allow for spatial and temporal control

In order to enter the cell cycle, it must

Regardless of whether its from G0 or G1 in order to enter the cell cycle again it must pass through the RESTRICTION POINT - molecular gate that blocks progression until nutrients and mitogens are present that suggest that proliferation is favored

Filament severing proteins

Sometimes its useful to create free barbed or pointed ends Gelsolin - 6 domains - domain 2 binds to the side of an actin filament, positions domain 1 to bind between subunits - caps barbed end, creates new pointed end ADF/cofilin - binds to ADP-actin subunits in filament - changes helical twist - strain causes it to break

Smooth muscle

Spindle shaped cells single nucleus homogeneous cytoplasm - not striated surround the intestine and aid in digestion

Class 3 of Actin crosslinkers

beta sheets - ABP 120 - Filamin/ABP

CDK and Cyclin Genes

multiple expressed in humans different cyclins and CDKS for different phases of the cell cycle

Second major point of regulation in the cell cycle occurs at

the G2/M phase transition

Critical role of actin in the cell

1) determine and change the cell shape 2) Forms cell cortex - excludes organelles - reinforce the plasma membrane - resist forces 3) Transmit forces - Promote dynamic cell morphology and motility occurs through polymerization and depolymerization of the cortical network - push membrane forward in pseudopods - phagocytosis (membrane out and around) Tracks for myosin motors - muscle contraction, organelle transport, cytokinesis

Duchenne muscular dystrophy

1/3500 males worldwide Progressive muscle degeneration - cardiac and respiratory failure in early 20s - cant resist the damage Most often caused by frame shift deletions in dystrophin Beckers is a more mild form - in frame deletions

Intermediate filaments

10-nanometer filament diameter - intermediate between the other 2 - larger than actin but smaller than microtubules No motors Used for mechanical support for cells IF subunits are many and varied - allow for a variety of functions - all tied to strength and resilience They all share an a-helical coiled-coil rod domaine at the core with variable N and C terminal head domains at each end

Normal cell cycle phases

4 different phases - a series of temporally controlled biochemical reactions G1 - most of the growth of the cell S phase - DNA replication G2 M phase - Mitosis and cytokinesis - generates 2 cells both heading to G1 A lot of Eukaryotes have this cycle

Nucleation thermodynamics

A monomer is favored until you get to a trimer the trimer has multiple interactions that stabilize it

Nucleation- Bacterial hijackers

A number of bacterial/viral pathogens can mes with actin polymerization machinery allows them to - move in the cell - spread to other cells - evade the hosts immune system EX.// Listeria

Class 2 of Actin crosslinkers

A-helical domains - A-actinin -spectrin/fodrin - dystrophin Forms contractile bundles

Are microtubules more similar to actin or intermediate filaments

ACTIN assembled from the same basic structural component in all cells intermidate filaments vary greatly

One of the most abundant and conserved proteins in eukaryotic cells

ACTIN up to 15% of total protein and greater than 90% sequence conservation

Checkpoints that must be turned On

ALl DNA damage pathways must be activated in the presence of problems with the DNA turned on by different signals by default they are off

Activation of myosin by CA in Skeletal muscle

AT rest troponin I-T complex moves tropomyosin into a position that interferes with myosin binding Calcium released during movement binds to Troponin-C this relieves the inhibition allowing for actin-myosin interactions

Mutations in DNA Damage checkpoints

ATM mutations - Ataxia Telangiectasia/ Louis bar syndrome - progressive ataxia - Dilated blood vessels of the skin - Immune defects ATR mutatations - Seckel syndrome - Postnatal dwarfism - microcephaly - intrauterine growth defects - retardation Transducer mutations - cancer predisposition

Cellular IF neurofilaments

Abundant in axons 3 types of subunits that form heteropolymers Level of neurofilament expression controls axon diameter - directly impacts nerve conduction velocity

3 filaments of the eukaryotic cytoskeleton

Actin - polymers made of actin monomer - main role is movement, adhering Intermediate filaments - Named based on size, also polymers (of multiple different protein) - Provide mechanical stability in the cell Microtubules - longest, stiffer - resist compression - main component of intracellular transport (tracks)

Which type of filament is best for motor protiens

Actin and microtubules They are polar and have a direction to them that the motor protein can use to orient itself and move.

The SCF complex (skp, cullin, F-box containing complex)

Activates CDKs in S phase Ubiquitinates the CKI inhibiting it - degrading the inhibitor - allows for activation

What drives cell cycle progression

Activation and inactivation of Cyclin-CDK complexes specific cyclins activating and deactivating CDK at specific times

Transitions between cell cycle phases are dependent on

Activation and inactivation of different Cyclin-CDK complexes

Two feedback loops in M-phase CDK actiation

Active M-CDK inhibits Wee1 from phosphorylating the active M-CDK earlier on - preventing its initial inactivation It Also increases the activation of Cdc25 to remove the inhibition from complexes already effected by Wee 1

Filament anchoring factors

Anchor actin to structures in the cell - plasma membrane for example ERM Family - Anchor actin filaments to the plasma membrane - allows morphology and mechanical stiffness to be effected Spectrin - forms a scaffold with actin filaments at cell cortex - ensures plasma membrane integrity - BLOOD CELLS SHAPE IQGAP - involved in cytokinesis - associates with plasma membrane and binds actin filaments - during actomyosin ring constriction in cytokinesis Dystrophin - connects actin to the membrane and extracellular matrix - critical to muscle strength

What happens to the kinetochores as Microtubules grow and shrink

As they grow and shrink (assemble and disassemble) They remained bound Due to many weak electrostatic interactions between - Ndc80, a + part of the kinetochore - the negatively charged microtubule surface

critical concentration (Cc)

Assembly and disassembly rates are equal the concentration where the addition and loss of a monomer occurs at the same rate (steady state) Is different at the + and - ends

CDKs are subject to complex regulation including:

Association with cyclins (regulatory subunit) Phosphorylation by CDK-Activating kinase (CAK) Inactivation by Wee1 kinase and Activation by Cdc25 phosphatase Association with CKI Subcellular localization (cytoplasm vs nucleus) - certain Cyclins in certain location Periodic degradation of cyclins and CKIs

Centrosomes role in the mitotic spindle

At the end of Mitosis each daughter cell has 1 centrosome This centrosome is duplicated during S phase - then move to opposite sides of the nuclear envelope with chromosomes between them This ensures the Bipolar spindle formation

What are checkpionts

Biochemical circuits that detect problems and send inhibitory signals to cell cycle machinery G1 (restriction point) - is the environment favorable for the cell to divide - (tissue growth and regeneration) S phase - is synthesis going poorly - are there mistakes - is the replication fork intact G2 - is all of the DNA replicated - is the DNA damaged - Is the environment favorable metaphase to anaphase - are all the chromosomes attached to the spindle

The cell cycle integrates

Both growth and division Interphase - growth and chromosome replication - where most growth is occurring - DNA replicated here Mitosis/meiosis - chromosomal segregation Cytokinesis - cell division - creates 2 daughter cells

Activation of myosin by CA in Smooth muscle

Ca binds to calmodulin which activates myosin light chain kinase this phosphorylates Myosin Light chain causes it to: allow binding site to interact with actin unfolds tail domain so the myosin can form into filaments then contraction occurs at 2 layers - one parallel - one perpendicular

How is the cell division completed

Cell cleaved into two new daughter cells in a process known as cytokinesis

Pioneering genetic screens in yeast yeilded

Cell division Cycle (CDC) Mutants Identification of cell cycle regulators relied on the use of conditional mutant - different phases have different morphologies

Loss of restriction point control leads to

Cellular transformations and cancer cells become overcrowded

kinetochore-microtubule attachments become stabilized When

Chromosomes pairs are bioriented - only stable orientation - thought to be due to tension stabilizing it some intermediates do form but are unstable

What can cause the control system to arrest in general

Completion of cell cycle events signals from the environment cell cycle is arrested at transition points (checkpoints)

Sarcomere

Contractile unit of the muscle made up of thick and thin filaments run from Z disk to Z disk The light bands are thin filaments Dark bands are thick filaments

How does the cell know when to separate the chromosomes

Controlled by the spindle assembly checkpoint Monitors the attachments between kinetochores and mitotic spindles (microtubules) prevents the metaphase to anaphase transition until the kinetochores are bioriented

Filament crosslinkers

Cross Linking requires proteins with 2 actin binding sites - typically dimeric allows formation of cables and increases there strength not designed for rapid movement - slow release three different classes based on what is being folded 1- EF hands - fimbrin /plastin 2- A-helical domains - A-actinin -spectin/fodrin - dystrophin 3- beta sheets - ABP 120 - Filamin/ABP

Cardiac muscle

Cylindrical shape up to 50 cm Long - typically much shorter multiple nuclei located near plasma membrane - less than the skeletal muscle make up the heart

Major activity of S phase

DNA replication But only once per cycle

Forming the Pre Replicative complex (pre RC)

DURING G1 Cdc6 binds with an ORC at the origin These bind helicase 2 copies of Helicase are added next to the DNA origin Forming the Pre Replicative complex (pre RC)

Regulation of the restriction point

Depends on a number of tumor suppressors or oncogenes Rb is a tumor suppressor gene - its inactivation leads to cancer - no prevention through the restriction site - cells pass through the restriction point even without external signals normally present Oncogenes - Hyperactivated forms of proteins that drive cells through the restriction site - Oncogene mutations can mimic the signal that sends the cell through another cycle

Chromosomes occupy

Discrete territories within the nucleus The location helps regulate its expression and transcriptional activity 3d organization is important for many genome functions - gene expression, DNA repair, DNA replication

Generic roles of cytoskeletal motors

Displace the cytoskeleton - convert energy from ATP hydrolysis to force that can move a fiber - often the motor is anchored - Think muscle transport cargo - convert energy from ATP hydrolysis to force that can move cargo along the fiber - motor is not anchored so the force just ends up moving it along the fiber - Most intracellular transport needs this mechanism sense tension - everything is anchored - the motor senses changes in tension or force that is dissipated as heat rather than movement

Drosophila cell cycle

Divide 13 times in 2 hours divide in synchrony - common cytoplasm - same biochemical signals going to all cells really only have S phase and M phase

APC activation

Done when all of the kinetochores are attached - No MAD 2 being signaled to inhibit APC APC ubiquinates several substrates in order to help the cell proceed from metaphase to metaphase - M-phase Cyclin A protein called Securin - This binds to and inhibits Separase - When degraded the separase is freed Separase - Proteolytically cleaves the cohesin complexes that link sister chromosomes together Allowing anaphase chromosomes to separate to opposite ends of the spindle

Changes that occur at the G2/M phase transition

Dramatic morphological changes occur The microtubule cytoskeleton is disassembled The mitotic spindle is assembled Chromosomes condense into discrete bodies the nuclear envelope breaks down Organelle systems are fragmented - golgi, mitochondria, ER Then they undergo mitosis and cytokinesis What causes these? activation of M phase cyclin-CDK complexes that lead to phosphorylation events

Filaments polarity

Due to the + and - end mechanics filaments have polarity with a Barbed - + end - grows quicker and has a higher association rate and Pointed - - end

When does nuclear envelope reformation Begin

During telophase Lanin dephosphorylation allows the nuclear envelope to initially reform around individual condensing chromosomes which are later used to form a complete nucleus

ACTIN

Dymeric helical structure - flexible filaments that can be cross linked into bundles or branched arrays - highly dynamic Main component of skeletal muscle - tracks for myosin motors Involved in increasing the surface area for nutrient absorption Sustain compression and tension Drive changes in cell shape

Orientation of Kinesin and Dyneine

Dynein is - end directed - massive enzymes Kinesin is + end directed - way smaller

Alpha and Beta tubulin and GTP

Each alpha and beta tubulin can bind to one GTP molecule In alpha tubulin the GTP is buried and never hydrolyzed In Beta tubulin it is exposed and can be properly hydrolyzed

Origin of replication

Established in G1 origin recognition complexes bind to DNA Pre Replication Complexes are established at the origin before the cell enters the restriction point

Nucleation of cables

FORMINS - homodimers assist in nucleating new actin filaments and remain associated with rapidly growing + ends as they grow Does so through its ability to bind profilin to the pointed end at a higher rate than normal - looks like tentacles

Cross linkers can dictate the type of actin bundles

Fimbrin makes parallel bundles when bound to actin - small - tight packing prevents myosin II from entering the bundle A-Actin makes contractile bundles - loose packing allows myosin to enter the bundle

Intermediate filaments

Flexible cables - often homodimers or heterodimers of long proteins that have alpha helical centers - form coiled coil interactions with one another Primarily add structural stability - form or maintain cell shape - mechanical support for cells, Prevents excessive stretching Sustain tension, NOT COMPRESSION Provides nuclear lamina architecture (Interact with chromosomes and effect gene expression)

Nucleation of branched filaments

Formation mediated by ARP2/3 complex 7 subunit complex (includes Actin related proteins 2 and 3 recruites a single actin monomer and mimic the trimer state - rate limiting step is overcome and growth is initiated - only the + end grows the other (pointy) end is capped Capped end causes branches to shoot out

What is the rate limiting step of actin filament assembly

Formation of a polymer nucleus (nucleation step) getting 3 monomers to interact with one another is unfavorable so it takes some time ( if you add preformed oligomers then it reaches stead state much faster)

Cellular IF- Keratins

Found in epidermal cells of skin Tough layer of cells that prevent desiccation and damage due to abrasion - different proteins are expressed in different parts of the epidermis Keratin networks form structural core of the dead (keratinized) skin layer Insert at desmosomes and hemidesmosomes - cell to cell - cell to matrix junctions MUTATIONS lead to cell rupture after slight stress - poor binding to the lower layers of the skin - seen as blistering and fragile skin

Centrosomes

Functions as the microtubule organizing center - initiating sites for MT assembly - most common type Have a pair of centrioles that recruit pericentriolar material - including G-tubulin ring complex Gets all the nucleating factors in one spot - all minus ends are linked on ons side whe

GTP-tubulin vs GDP- tubulin

GTP - packs efficiently - high concentration leads to growth - hydrolysis to GDP GDP - 100x faster depolymerization - higher concentrations causes the tubule to dissociate and shrink

Example of a checkpoint pathway

Genotoxic stress - DNA damage - activators sensors Sensors - ATM, ATR - Activate via phosphorylation transducers Transducers - Kinases, transcription factors - Act to alter cell cycle regulation and DNA repair - Leads to cellular responses Responses - cell cycle arrest - DNA repair - apoptosis

Evidence of astral stimulation model

Glass beads displace the spindle to one side of the cell this causes a furrow to form on one side of the cell - leads to an incomplete cleavage - creates a binucleated cell At next mitotic separation cleavage occurs both between the centromere linked by mitotic spindles but also the ones adjacent to it suggests the astors induce cleavage

How do molecular motors move along filaments (theories)

Hand over hand - two dimeric heads take turns being the lead - step over one another -( proved by labeling one and measuring the step size, if every other step then it is around 16 nm) Inch worm - always in the same order - stretch , then slide back together - repeat

g-tubulin ring complex

Helps get over the rate limiting step and has a stabilizing effect on the microtubule binds 7 Alpha betas dimers and generates a spiral that starts off the microtubule acts as a template basically

Cellular IF- Nuclear Lamins

IF that surround the nucleus 2 different genes - A and B Form dense network that encases the nucleus Also seem to affect chromatin orginization and gene expression - mutations lead to a wide variety of disorders This is broken down and reformed during cellular division - Broken down through phosphorylation. They can no longer form a tight network

Rho family GTPases

Important regulators of actin assembly Family of small GTPases similar to rands Actrivating RAC - leads toi branched web formation - less stress fiber formation Activating ROH - Increased myosin activiy - more stress fibers By upregulating different GTPases - certain actin filaments are promoted

CDK inactivation

Inactivated through further phosphorylation Important for G2/M phase regulation or Direct Binding of Cyclin-dependent kinase inhibitors (CKIs) Bind directly with the complex and keep the complex inactive Important for G1/S phase regulation

The anaphase promoting complex/cyclosome

Inactivates CDKS in M-phase by ubiquitinated cyclin B

Where is myosin in the rigor state

It is also known as the attached state There is no ATP bound and the myosin is attached to actin Happens during death because there is no ATP to pull it off.

Microtubule severing Enzymes

Katanin Spastin Fidgetin members of AAA ATPase family often hexamers of ATPases that bind to the surface (c-terminal tails) and remove tubulin dimers from the middle of the lattice making it fall apart.

Phosphorylation of lamins and nucleoporins

Lamins are intermediate filaments that make up the nuclear lamina - when phosphorylated they are broken down Lead to nuclear envelope disassembly Phosphorylated by M-cyclin CDK complexes

Steps to kinetochore Microtubule attachment

Late Prophase - Mitotic spindle pole are moved to each side of the cell Following Nuclear envelope breakdown Sister chromatid pairs are exposed to a larger number of + ends of microtubules from the spindle poles - Minus ends are bound to centresomes Kinetochores are first attached to the side of the microtubules - At the same time pushing the arms of the chromosome outward - this prevents blocking the kinetochore and allowing microtubules in Microtubules eventually bind to kinetochore at the end instead of the side - they are then captured and stabilized When both poles stabilize this creates Bi-orientation - 2 kinetochores are bound to microtubules from opposite poles

DNA damage in G1

Leads to inhibition of G1/ CDK activity A break occurs various kinases are recruited to the site of damage - initiates pathway leading to cell cycle arrest Chk1/2 phosphorylate p53 P53 - interacts with E3 ubiquitin MdM2 - When its phosphorylated it can't interact - Levels rise with damaged DNA because of this Phosphorylated P53 is now stable and active - binds to regulatory region of P21 gene P21 - Is a CKI (cdk inhibitory protein) - High levels inactivate G1/S-CDK PREVENTS the cell to progress from G1 into S or S into G2 Similar checkpoint pathways control Cdk Activities during other cell cycle stages

Structure of Kinesin

Lots of different versions - 45 genes encode them - allows for specific function Head chains of the motor contain the catalytic site - at N terminus Tail used for dimerization and binding cargo - light chains are used to select cargo Middle is a coiled coil structure Similar to myosin in structure*

Microtubules

Made of Ab-Tubulin dimers - expressed in different genes but always found together. The a and B tubulin form end to end creating a protofilament usually 13-15 protofilaments form a tube - but the protofilaments are formed in a spiral pattern - not created then assembled into a tube One end terminates in an alpha and the other in the beta - it is polar - also has a + and - end + end is fast growing (beta end) - end is slow growing (alpha end)

STRUCTURE OF INTERMEDIATE FILAMENTS

Made up of multiple varied subunits - allows for variety - variety allows for different functions all tied to strength and structure They all have a helical coiled coil rod domain at the core Variable N and C terminal head domains at each end

epithelial cells cytoskeleton

Micro villi -formed from actin cables pushing the membrane out ACTIN web - at both apical and basal side of cell - help prevent organelles from moving out of the cell and provide some support Intermediate filaments provide additional support - link to desmosomes that connect cells together or hemidesmosomes that connect the cell to basal lamina - resist stretching forces on cell Microtubules act as tracks - move things from one side to the other

Organization and Function of the mitotic spindle is dependent on

Microtubule based motors Kinesin and Dynein Motors play 2 roles Kinesin - Cross link to stabilize length Dynein - Properly position The spindle and chromosome (some kinesin also positions chromosomes

Restriction point control basic steps

Mitogens activate immediate early genes Cyclin D is expressed CDK-CYclin D inactivates RB EF2 is now active and induces G1/S regulator expression + feedback loops further inactivate Rb and activate E2F Cells are committed to replicating DNA and progressing

Mitogens simple pathway

Mitogens trigger passage through the restriction site and promote cellular proliferation Does so by activating immediate early gene expression Immediate early genes include - The transcription factor MYC Myc - increases expression of delayed response genes - Promote expression of G1 cyclins ESPECIALLY Cyclin D

The restriction point

Molecular gate that regulates expression of genes required for cell cycle progression In the presence of proliferation signals from the surrounding environment -Growth factors - extracellular matrix The cell wants to exit G1 and commit to another round of proliferation In order to do so it must pass through the restriction site

Intermediate filament assembly

Monomer has an N and C terminal end with an alpha helical region between them these then form homo or heterodimers in a COiled-coil fashion Dimers then bind slightly offset to form a staggered tetramer Tetramers then associate together forming a filament which can grow in these sections

Actin Basic properties

Monomer is 375 amino acids long composed of 2 similarly folded domains binding pocket for MG-ATP/ADP - WHen ATP is bound it is slowly hydrolyzed Essentially all monomers are ATP bound in VIVO Monomers interact head to tail to form double stranded helical filaments

actin polymerization

Monomers interact head to tail to form a double stranded filament Nucleation requires 3 interacting monomers reversible addition of ATP-actin monomers to each end

Structure of Dynein

Much larger than Kinesin Six triple A domains - only the first 4 can bind to ATP - Only AAA 1 is the catalytic site and controls conformational changes Long stalk and small globular microtubule binding domain coming off the end of it - not in same part of the molecule as the ATP binding and Microtubule binding Intermediate chains and light chains form large assembles to help bind to cargo as well Dynactin helps keep it attached to microtubules and vesicles

Condensin

Multiprotein complex - ASSISTS IN CHROMOSOME CONDENSATION and maintaining structure activated by CDK - phosphorylates the CAP_D - allows it to bind to DNA These look similar to cohesin complex Uses ATP hydrolysis to alter DNA structure

KERATIN DISORDERS

Mutations in 18 different keratins - each one results in fragile cells EPIDERMOLYSIS bullosa simplex - 1: 50 K - mutation in 2 keratin genes (k5 and K14) - Minor physical trauma results in rupture of basal cells in the dermis (causes blistering)

Laminopathies

Mutations in laminA Hutchinson-gilford Progeria syndrome - rare - premature aging - shortened stature, craniofacial disproportion, thin skin, alopecia, osteoporosis Average age of death 13 - greater than 90% due to atherosclerosis Caused by point mutation in Lamin A - causes deletion of 50 AA from tail - reveals cryptic splice site

Cohesinopathies

Mutations in the cohesin Ring and cofactors Cornelia de Lange Syndrome (dominant) Roberts syndrome (recessive) - limb abnormalities - abnormal growth Likely due to cohesins role in regulating gene expression

Intracellular transport by myosin

Myosin 5 moves toward + end Myosin 6 near minus end similar mechanism with the lever arm BUT it has 2 headgroups (dimeric) it leads to a walking motion carrying the cargo along

Direct evidence that myosin moves actin filament

Myosin heads are placed on a glass slide Actin is added to the myosin heads in the presence of ATP the myosin can move toward the plus end of the actin

Three major classes of cytoskeleton motors

Myosin, Kinesin and dynein all derived from primordial (ases) myosin and Kinesin are part of the GTPAse family Dynein is part of the AAA ATPase family

Actin polymerization phases

NUcleation (lag) - actin subunits bind together - requires 3 monomers Elongation - monomers are added to both ends to extend the filament Steady state - assembly and disassembly rates are equal. - coming on just as fast as falling off - Concentration at steady state is known as the critical concentration

Is an intermediate filament polarized

No once you get to a tetramer both ends are basically the same one of the reasons motors don't work on them - they need to know which way they are going

Retinoblastoma (Rb) and E2F

Normally bound together - RB inhibits the activity of E2f RB can be phosphorylated by Active Cdk-Cyclin D complex - Phosphorylation frees up the E2f E2F - can now freely function and transcribe Cyclins for G1/S and S phase Genes. - can also induce transcription of itself for a + feedback loop Active products from E2f when bound to CDK can go back and phosphorylate more RB and free up more E2F - Generating a + feedback loop These loops provide a point of no return.

Rho and RAC in neutrophil

On the receptor side where it is " seeing" the bacteria - RAC dominates - causes polymerization and protrusion On the other side - RHO dominates - Actin-myosin contraction

Active CdK-Cyclin D complex

Phosphorylate retinoblastoma protein to drive expression of S phase regulators Retinoblastoma is inactivated when phosphorylated - E2F proteins can now function and transcribe Cyclins, and a number of S phase genes E2F products create feedback loops freeing up more E2F and ensuring progression into S phase

Plus and Minus end of actin

Plus END - Grows faster than the - end. - fresh ATP-actin on this side compared to the - end Minus end - shrinks faster, (grows slower) - net disassembly - more ADP-actin than the + end leads to treadmilling - growth at one end, loss at the other - not going anywhere CAN HAVE DIFFERENT CRITICAL CONCENTRATIONS

Orientation of Actin in a sarcomere

Plus side is bound to the Z disk - CAP Z minus is toward the m line free - Tropomodulin caps the minus end

Mitogens Pathway

Proliferation signal Can bind to cell surface receptors to initiate intracellular signaling pathways RAS - major GTPase pathway - Activates map Kinase cascade Map kinase Cascade leads to increased expression of immediate early genes - Including MyC MyC - increases expression of delayed response genes - Including those that lead to INCREASED g1-Cdk activity - CYCLIN D being a major one

CDK phosphorylation effect on GolGI

Promote organelle system disassembly prevents vesicle fusion into golgi stack leads to lots of vesicles

The assembly of the Contractile ring

Promoted by Small GTPase RHO A this has 2 major roles Activates formin - promotes assembly of actin filaments Promotes phosphorylation of Myosin light chain - activates myosin activity

Protein degradations effect on CDK

Provides both Positive and negative regulation to CDKS Degradation of the cyclin subunit will lead to inactivation CKIs are degraded as well leading to activation of the complex

Microtubule-associated proteins (MAPs)

Regulate MT dynamics in cells have stabilizing and destabilizing effects on either end of the tubule

Cyclin-dependent Kinases

Regulate cell cycle progression by modulating the activity of other proteins via phosphorylation Regulated by Cyclins - synthesised and degraded at specific times during the cell cycle - function as a cell cycle clock Cyclins dictated the level of activity of CDKs during specific points of the cell cycle

Cyclin-dependent Kinases

Regulate cell cycle progression by modulating the activity of other proteins via phosphorylation Regulated by Cyclins - synthesised and degraded at specific times during the cell cycle - function as a cell cycle clock Cyclins dictated the level of activity of CDKs during specific points of the cell cycle LEVELS OF CDK do not Change during the cell cycle - only the levels of the cyclins to activate them

How does DNA only replicate once in S phase

Regulatory mechanisms within S-Phase Cdc6, ORC, Cdt1 are phosphorylated by S-CDK and inactivated - preventing assembly of new replication complexes CDT1 is also sequestered by a protein called Geminin - this protein is present until the end of mitosis MCM proteins dissociate from the chromatin S phase Cyclin/CDK activity is downregulated after replication

Accurate segregation of chromosomes

Required to maintain genomic integrity Normal is 2 pairs of replicated chromosomes -this means 4 copies of Chrom 1 are present - corresponds to 4N DNA content DEFECTS during mitosis - cause aneuploidy or an abnormal amount of chromosomes - hallmark of cancer cells

ATP and actin

Reversible addition of ATP-actin monomers occurs at both end After polymerization - subunit conformation changes and ATP is hydrolyzed ADP-actin dissociates more readily from the filament than ATP-ACTIN

Microtubules

Rigid hollow Rods - heterodimeric (a and B) tubulin dimers add end to end forming protofilaments - protofilaments form a hollow tube RATHER STIFF - resist compression and tension Highly dynamic Form tracks for kinesin and dynein motors heavily involved in mitosis -organelle and chromosome transport

S phase DNA replication

S-CDK stimulates the assembly of several initiator proteins DDK phosphorylates helicase subunits - Activating them As DNA unwinds DNA polymerase and replication proteins come join and replicate DNA ONLY ONCE

Filament stabilization

Side Binding filament stabilizers - extended proteins that bind along the side of filaments Tropomyosin (tm) - increase tensile strength - Ca sensitive regulator of actomyosin interactions in muscle - in non-muscle cells allows filament to discriminate between other actin binding proteins

What specifies the location of the contractile ring formation?

Signals from the poles and central spindle specify its location Astral stimulation model - astral microtubules specify where the cleavage should occur Central spindle stimulation model - Factors located at the central spindle in the overlapping microtubules specify where it should assemble - RHOA activators localize to the central spindle Astral Relaxation Model - Astral microtubules inhibit contraction away from the center of the cell All three models are supported by evidence and likely vary by cell type

Kinesin Mechanochemical cycle

Similar to myosin Opposite affinity in terms of ATP and ADP ATP has a strong affinity for the microtubule - ADP form releases and steps over Neck linker docking produces a conformational change in the kinesin dimer

Cillia and FLagella

Specialized dynein and microtubule powered structures have a unique 9+2 arrangement - center has normal microtubules - 9 on the outside have A FULL and partial microtubule in each one Tails of dinene are anchored to the tail of the microtubule which is linked to another microtubule - sliding is prevented and force is translated into a BENDING MOTION

Microtubule Dynamic Instability

The concentration of tubulin is above the critical concentration for GTP-tubulin to assemble BUT not high enough for rapid growth to occur leads to competing hydrolysis of GTP and addition of new GTP REsults in Random rapid fluctuations in length - Shrinkage occurs when GTP-Tubulin (gtp cap) at + end is lost leaving exposed GDP tubulin - RESCUE phase then starts again by adding new GTP ccap and growing back out WHY? Allows MT to sample there surroundings - if it likes what it sees it stabilizes

The Primary force to move chromosomes around within the spindle and align them in the kinetochore is

The forces generated from disassembling the kinetochore microtubules

Myosin Structure

The head domain is highly conserved between all of the myosin genes - once past the head they can vary greatly Myosin has an Actin Binding site, Active site and 2 addition light chains for stabilization Actin binding site and active site are on opposite sides of the head

Nuclear subdomains (nuclear Bodies)

The nucleus contains subdomains called NUCLEAR BODIES Non membrane bound organelles - used for specific biochemical processes Proteins can rapidly exchange between nuclear bodies Highly Dynamic

After DNA is replicated

The sister chromosomes are attached by Cohesin Cohesin - 4 subunit complex - Smc1 and 3 are coiled coil proteins with an ATPase domain - SCC3 and 1 connect the ATPase head domains forming a ring (needed to bind to DNA) This ring circles the sister chromatids - stay together until there is a signal to separate

Checkpoints that must be turned OFF

These are preventing the progression all the time - always on They must be satisfied before the progression can continue - they are then turned off Includes - Favorable Environment - Chromosome unattached to spindle

Why have 2 + feedback loops related to E2F

These loops provide a point of no return when leaving the G1 phase There is no way for them to go back because the signal to progress will now signal itself

Muscle cell orginization

Thick myosin filaments and thin actin filaments make up sarcomeres and the myofibril the myofibril come together to make a muscle fiber

Filament Capping

Through Cappers bind to either barbed or pointed end of filament to block subunit addition or dissociation ( can be blocked on the barbed end by formin but not ARP2/3)

How Does the cell know when to inactivate or activate CDKs?

Through the help of checkpoints they detect problems or changes and sends signals to the cell cycle machinary

Cell division always occurs

Through the middle of the spindle How is this location defined?

Mechanisms that control cell cycle transitions compared to other cells

Timings may be different Cell cycle lengths may vary the mechanisms that control the transitions between the steps are very similar

Basic anatomy of DNA damage checkpoints For G1/S aand G2/M

Top to bottom Genotoxic stress - problem with replication fork - break in the DNA Sensors - mostly kinases (ATM and ATR - recruit other proteins (transducers) Transducers/ Effectors - Triggers effectors - Act directly on Cell cycle proteins (CDKs) - Halt the cell cycle or upregulate DNA repair - cause a response Respone - DNA repair - Cell cycle Arrest - Apoptosis if necessary

Activation of M-Phase Cyclin-Cdk complexes

Triggers the morphological changes seen at G2/M transition M phase cyclin interacting with CDK - forms inactive complex Cyclin CDK complex must then be activated by CDK activating Kinase (CAK) - Forms active complex Wee1 phosphorylates this complex at the same time as CAK - causes inactivation of the "activated" complex - kept inactive until conditions for mitotic entry are met Cdc25 phosphatase - opposes activity of Wee 1 once the balance of kinase and phosphatase activity tips toward Cdc25 a large pool of m-CDK is activated - this leads to positive feedback loops to keep it on this path Cell is COMMITTED TO mitosis

Anti CDK drugs

Tumors grow without normal cell cycle control Idea is to inhibit CDKs to prevent transitions from steps in cell cycle - original ones showed toxicities due to poor specificity Dinaciclib - non specific - multi-CDK inhibitor - blocks some but not all Palcicilib - Specific inhibitor for CDK 4/6 - Main G1 CDKs in humans - Used for certain breast cancers KEY Specificity and patient selection is key - certain mutations make them either great or useless.

Protein degradation through ubiquitination

Ubiquitin- enzymes add a small peptide (ubiquitin) to the target - chains of these tags mark the protein for degradation through the proteasome Ubiquitin is trimeric E1 ( low abundance) - activates the ubiquitin - transfers to E2 E2 - Complex with E3 - or transfer ubiquitin to E3 E3 (high abundance, high specificity) - Complexes with E2 to provide substrate specificity and directly transfers ubiquitin to target - transfers the ubiquitin tag to the target THe proteasome recognizes these tags and degrades the protein

How is kinetochore attachment recognized

Unattached kinetochores generate a diffusible signal that prevent cells from making the metaphase to anaphase transition It is a necessary wait signal - if destroyed (with a laser) the cell will go on like it had been stabilized - This wait signal is known as Mad 1 and 2 MAD 1 and 2 - bind to kinetochore - it recruits other Mad molecules that go onto target APC or Anaphase promoting complex (an E3 ubiquitin) inhibiting it

Regulation of contraction in skeletal muscle

Voluntary accomplished by inhibiting or allowing myosin binding to actin - normally inhibited by troponin blocking the interaction between myosin and actin Calcium removes the inhibition and allows the myosin to interact with actin - troponin and tropomyosin move

Affinity of Myosin bound to ATP

Weak affinity for actin When ATP binds it causes the head group to fall off as its hydrolyzed it moves ADP has a high affinity for actin the power stroke removes the ADP and moves the head

Wee 1 and Cdc25

Wee1 kinase - inhibitory - quickly turns off the CDK - Phosphorylates it just above the ATP binding site preventing hydrolysis Cdc25 - Phosphatase - removes inhibitory phosphate IMPORTANT FOR G2/M regulation

Wee 1 and Cdc25 in activation of M-phase CDK

Wee1 phosphorylates the Cyclin-CDK complex at the same time as CAK - Keeps the activated CDK inactive CDC25 is dephosphorylated the inactive CDK complex allowing it to activate two competing forces Once the Cdc25 starts to win the Active M-CDK triggers 2 feedback loops to prevent it from going back into g2 and commit to Mitosis

CDK activation

activated through cyclin binding and phosphorylation of the T loop Partial activation through cyclin binding Full activation through CDK-activating kinase

Kinetochores

assemble at centromere Allow chromosomes to attach to spindle microtubules each one can bind to multiple + ends of microtubules

Anti parallel microtubule sliding

by kinesin 5 and 14 - move towards minus ends + ends in opposite directions so they help pull them back together after they move apart

DNA damage checkpoint

can inactivate cell cycle progression at numerous stages in the cell cycle

cycling time between different cells

can vary significantly Drosophila undergo 13 divisions in 20 hours

Tissue contains

cells in a variety of proliferative states Stem cells divide due to environmental signals - differentiate into different cells based on necessary signals - Divide to form another stem cell and one cell that goes to be differentiated

Dystrophin

connects actin to the membrane and extracellular matrix - critical to muscle strength Located on X chromosome - Huge protein and gene - large target for mutations

What is one of the main things that must be accomplished for the cell to move from G1 to commit to another round of cell cycle

degrade these CKIs around to allow activation of certain CDKS specific to G1/s phase

Efficiency of Tubulin packing

depends on GTP/GDP state GDP tubulin bind less strongly to each other - depolymerization is 100X faster from ends with GDP-tubulin Whether a given end grows or shrinks depends on rate of GTP-tubulin addition vs GTP hydrolysis

Cytoskeletal structures undergo

dramatic rearrangements to serve different purposes During cell division everything is disassembled and reassembled into a different structure then reorganized back into proper structure THE STRUCTURE IS STRONG BUT FLEXIBLE ENOUGH TO CHANGE WHEN NEEDED

Mutations in genes that regulate cell cycle progression (yeast)

drastically change cell morphology in yeast

Cytoskeleton filaments are important for

establishing and maintaining cell shape and polarity

The mitotic spindle

facilitates chromosome segregation Bipolar - ensured through nucleation from 2 centrosomes Composed of microtubules - made of tubulin dimers - 13 protofilaments - Fast growing + end with a slower growing - end Spindle assembly requires spatial and temporal control of microtubules dynamics

Class 1 of Actin crosslinkers

fimbrin /plastin Bind EF hands Forms tight parallel bundle

Formins vs ARP2/3

formins bind at the plus end and prevent other regulators from working - stays bound - cables get very long binds to pointed end and caps it and releases - branches tend to be shorter - more like a mesh of capped filaments

T-loop

found in the CDK folded over and inhibits the ATP binding pocket of the CDK enzyme When cyclin binds there is a conformational change and it moves out of the active site partially CDK-activating kinase - phosphorylates the T-loop and moves it completely out of the way of the binding pocket

Rate limitng step in Microtubule formaion

getting 13 alpha beta tubulin dimers assemble at one time proteins help with this by binding to the minus end of the tubules Gamma tubulin forms dimers and creates a spiral structure that starts things off essentially

Rearrangements of the cytoskeleton

have to occur rapidly for some functions WHite blood cells - The actin in front responds to the chemoreceptors as it chases the bacteria - this actin is constantly rearranging to "chase" the bacteria

The cell spends most of its time in

interphase - most of the 16-20 hour life division occurs rapidly

Skeletal muscle

long Cylindrical shaped cells -up to 50 cm long - formed through fusion of multiple precursors multiple nuclei located near plasma membrane also known as striated muscle

Kinesins and Dynein are important for

long range transport in mammalian cells Most extreme example is transport along the axon of a neuron - cell body to nerve terminal Anterograde = to nerve terminal - Kinesins Retrograde = back to cell body - Dyneins

How were kinesins discovered

looking at anterograde path of neurons

Nucleation of filament growth

occurs in 2 ways Nucleation leading to a meshwork of branched actin filaments - cell cortex Nucleation leading to straight parallel bundles of actin (cables) - Microvilli

How long is a myosin head interacting with actin

only about 5% of the cycle this allows it to freely interact with other heads The myosin stays stationary during the power stroke and leads to a shortening of the fiber as it pulls actin towards the center

Mechanochemical cycle of myosin

requires ATP to move Myosins conformation and affinity for actin changes based on charged state ATP binds to myosin - dissociates from actin ATP is hydrolyzed - ADP is bound to myosin - Lever arm COCKS and moves the head group ADP bound myosin - binds to actin - has high affinity for actin ADP is released - known as the power stroke - the head snaps back to its original conformation - this motion causes movement of the actin

Actin binding proteins (types)

sequestering nucleation capping stabilization crosslinking severing anchoring motors

What is the cell cycle

series of events that allows a cell to duplicate into two cells series of biochemical reactions where transitions from one phase to the next require assembly of new enzymes and inactivation of old enzymes - His definition

Cytokinesis depends on

the assembly of an actin myosin ring at the center of the spindle Myosin recruited during anaphase - activated after separation of chromosomes Ring contraction pulls the membrane in to form 2 daughter cells - requires a net increase of membrane surface area - this requires the secretory vesicles of the golgi

Entry into the cell cycle is controlled by

the restriction/ start point -- molecular gate that blocks progression until nutrients and mitogens are present that suggest that proliferation is favored

Bacteria cytoskeleton

there are ancient homologs for all three filaments functions are conserved across all life FTZ - tubulin homolog MreB - ACTIN - important for cell shape and rod factor ParM - also actin - segregation during replication

+tip proteins

track growing or shortening microtubules Preferentially bind to growing MT Plus ends - due to increased affinity for GTP-tubulin can either promote addition or removal of A-B tubulin dimers can directly modify the microtubule dynamics

Experimental Measurements of Kinesin Stepping

used to test how it moves along the tubule model was tested using optical trapping and TIRF microscopy Motor is attached to a bead Bead is held inside a focused laser that applies a resistive force as it tries to walk Each step can be seen in the data (if you only label one head then you can determine if it is inchworming because there would be larger steps seen)

Most of our knowledge about the cell cycle comes from

yeast fission yeast - all steps of the cycle budding yeast - missing G2 phase - buds off of mother cell Great to study because - simple - replicate fast - eukaryotes - change morphology as they go through the cell cycle