Module 5

Question 1

What is the cytoskeleton

Answer 1

-a network of structural proteins that are found in all cell types
-this filamentous array occupies large portion of cytosol and expands through the cytoplasm from organelle to organelle and to the plasma membrane
-permits signalling and vesicular transport
-defines shape and distribution of cellular contents
components: microtubules, nucleus, actin filaments
three classes of structural proteins within:
intermediate filaments: add mechanical strength to cell
microtubules: support trafficking within cell
actin: support cellular motility or other large-scale movements like contraction

Question 2

Cytoskeletal protein functions

Answer 2

Binding- bind to a target like similar proteins to form polymers (molecule made of a large number of repeating molecules)
Conformation- when cytoskeletal proteins binds, they undergo conformational changes
Function- function of proteins defined by the number and type of cytoskeletal proteins that are bound

Question 3

Detailed summary intermediate filaments

Answer 3

-supply strength to cells to allow them to resist change (ex. through absorbing stress)
-strongest filament
-polymers and their expression is tissue and cell specific
-assembly/disassembly controlled by post-translational modifications of individual protein
-organized spontaneously and not very organized

Question 4

Major classes of intermediate filaments

Answer 4

-reflective of cell needs
-organized based on protein type, dictating their distribution and function
-class 1 and 2 keratins most common in humans

class I protein: acidic keratins, Distribution: epithelial cells, Proposed function: tissue and strength integrity

class II: basic keratins-epithelial cells-tissue strength and integrity

class III: desmin, GFAP, vimentin, periphevin-muscle, glial cells, mesenchymal cells, perphevin neurons- sarcomere organization integrity (organizational part of muscle)

class IV: neurofilaments-nurons-axon organization

Class V: lamins-nucelus-nucelar structure and organization

Class VI: Phakinin (CP49), Filensin- lens-specific beaded intermediate filaments

Question 5

what determines strength of intermediate filaments

Answer 5

-how the individual proteins are packaged and assembled into polymers
-dont gain their properties till assembled
-at beginning just primary structure with a polymer of amino acids linked with peptide bonds and at this point not stronger than any other protein

strength come from secondary structure
-can be a-helices, beta-sheets, and random coils
-very rich in a-helicies, giving some protperties like long, coiled structure of filaments and the hydrogen bonds stablize the structure as resist the stretching of filaments and prevent it to collapse

Question 6

what is a-helices

Answer 6

a tight coil that forms hydrogen bonds with the backbone of every fourth amino acids

Question 7

what is a beta sheet

Answer 7

planes are formed between rows of amino acids with hydrogen bonds between the backbones

Question 8

Structures of intermediate filaments

Answer 8

Monomer
-coiled monomer=tertiary structural level
(2 little pick wavy lines chillin beside eachother, one end reads NH2 the other end reads COOH

Dimer
-2 coiled monomers come together by wrapping around each other
-formed coiled coil
-that structure allows for maximum contact (hydrogen bonding) between two peptides and thus conveys tremendous strength
-quaternary structure
(wrapped as if knitted blanket)

Tetramer
-2 dimer assemble in antiparallel ( NH2 and COOH termini on opposite ends) staggered manner
-since dimers aligned lengthwise, hydrogen bonding adn future strength have increased
-fundamental building block
(the dimers unevenly and reversely laying on eachother)

Now tetramers come together in 3 stages to form filaments

1. formation of unit-length filaments
   -8 tetramers coming together
   -height ground to sky is 20 nm

2.formation of immature filament
-bunch of unit-length filaments coming together
-interact loosely end-to-end
-height sky to ground is 20 nm

3. formation of mature filaments
   -immature filaments compact
   -now fully assembled intermediate filament
   -height sky to ground is 10 nm

Question 9

post-translational modifications for intermediate filaments

Answer 9

-control shape and function
-main types: phosphorylation (addition of phosphate groups) which leads to dissolution of intermediate filaments into unit-length filaments and when removed by enzyme called phosphatases, intermediate filaments spontaneously reform and glycosylation (adddition of sugar groups)
-typically occur in head and tail domains
-phosphorylation assembly and disassembly important for cell division

Question 10

Specialized intermediate filaments

Answer 10

Lamin
-solely in nucelus and forms the nuclear matrix, a dense network to protect chromatin

Desmin
-does not form long, thin filamentous structures but more so connects different cellular structures together
-important for muscle structural integrity

Keratins
-binds desmosomes to form a complex
-makes up hair, skin and nails

Question 11

A detailed summary of microtubules

Answer 11

-primary purpose- cellular trafficking (movement of proteins, vesicles and some cellular organelles within the cytoplasm)
-microtubules defined how things trafficked through cytoplasm with purpose with specific routes cargo can go
-can be bi-directional travel along single microtubule and cargo can attach or detach anywhere along length
-determine where things move within cell
-can create or remove routes

Question 12

organization of microtubules

Answer 12

-organized
-assembly requires numerous proteins
-assembly occurs in regions called microtubule-organizing centers (MTOCs) (cellular structure from which microtubules arise)
-assembly different locations within cell

Question 13

MTOC example

Answer 13

-centrosome
-during cell division copied so that two resulting centrosomes can form poles of the mitotic spindles

Question 14

Protein structures of microtubules

Answer 14

-made of specific proteins called tubulins which represent a lot of cellular proteins which variety of functions
-composed of dimerized proteins

-care about tubulins a-tublin and beta- tublins
-both gobular with similar shapes who bind tightly together in a head-to-tail fashion to form dimer
-both bind to GTP molecule and beta-tubulin can cleave GTP to GDP
-when bound to GDP, beta-tubulin has shape change

Question 15

Microtubule polymerization

Answer 15

-formation from a- and beta tubulin dimers very dynamic
-if polymer made from individual tubulin dimers, can reach critical length and will continue to grow

dimers form polymers
-dimers spontaneously assemble into unstable polymers that can quickly fall apart

polymer growth
-once polymer of at least 6 dimer subunit forms, more stable and may grow laterally or longitudinally
-this is a protofilament

protofilament tubes
-protofilaments form sheet and assemble into tube of 13 protofilaments
-this the nucleation site for microtubule elongation
-even in tubular form, microtubule in dynamic state of assembly or disassembly

assembly/disassembly
-at ends of microtubule, dimers continue to come and go
-if rate of assembly greater than diassssembly, growth occurs
-rate of diassembly greater, microtubule shortens

Question 16

Key characteristics of microtubules

Answer 16

Assembly
-recall: a-tubulin has GTP bound where beta-tubulin can have GTP or GDP
-when GTP bound to beta-tubilin -> dimer polymerization is favored and dimers attach to each other

Disassembly
-beta-tubilin GTP is hydrolysed (chemical rxn in which molecule of water breaks) to GDP -> dimer undergo a confirmational change which promotes
depolymerization

Polarity
-as microtubules formed by end-to-end polymerization of dimers, ends are different and have polarity ( plus and minus end)
-prefered growth rate at plus end

Question 17

Microtubule dynamic instability

Answer 17

-need to change for cellular environment as numerous different spots and jobs etc
-Ability to grow or shrink

GTP cap
growing microtubule has cap of GTP subunits at its tip

Hydrolysis
GTP hydrolysis occasionally exposes GDP-bound subunits at the tip

Depolymerization
-following hydrolysis, rapid catastrophic depolymerization occurs

recap
-enough GTP subunits bind at once to recap microtubule and stop depolymerization

growth
-microtubule resumes growing when GTP- bound dimers are availible until another change in cellular environment detected

Question 18

explain catastrophe during microtubule dynamic instability

Answer 18

GTP converted to GDP on tubulin dimers at one end, they will fall off
-initate catastrophe
-which is rapid depolymerization of tubulin dimers at plus end, shortening microtubule

Question 19

measures against catastrophe during microtubule dynamic instability

Answer 19

-not unstoppable or permantent

Aversion: capping (haulting)
-once desired length, plus end bound to capping proteins
-add stability and keep them polymerized even if dimers are in GDP bound form

Reversal: Rescue
-can occur spontaneously if enough GTP-bound dimers present or occur in presence of another protein

Question 20

What advantages does dynamic instability of microtubules offer?

Answer 20

1. allow cell to explore cytosol rapidly and create new pathways for trafficking depending upon cells constantly changing needs
2. allows cells to exert force. Any molecule attached to microtubule near plus end will be transported through cell as microtubule grows or shrinks

Question 21

Microtubule associated proteins (MAPs)

Answer 21

-other protein roles: stability, cross linking, bundling, cutting

capping protein
-stabilize

rescue-associated protein
-stop microtubule catastrophe

motor proteins
- proteins control trafficking while microtubules control where molecules can go
-bind to cargo that need trafficking ( tails) and bind to microtubule and walk along it (heads)
-walking consumes cellular energy in form of ATP
2 main types (both heads contain microtubule-binding domains
1. Kinase
-move along microtubules towards plus end
tail- light chain
head-> stalk (coiled) -> tail
2. Dynein
-move towards minus end
tail- intermediate chain/light chain complex)
stalk -> head -> stem -> tail

Question 22

Walking of motor protein

Answer 22

-energy-dependent

Step 1
-Head 1 bound to microtubule and head 2 bound to ADP

step 2
-walking movement initiated by ATP binding to head 1, which causes a confirmational change that includes head 2 swinging around

step 3
-once head 2 over a binding site, binds to microtubule and releases ADP

step 4
-ATP head 1 then undergoes hydrolysis so its now ADP bound to head 1, which causes release from microtubule

step 5
-repeat but ATP now binding to head 2 causes head 1 to swing

Question 23

quick fast actin filaments summary

Answer 23

-has own motor proteins which it can bind to that initiate movement
-composed of globular proteins
-mostly move through cells itself as form stronger network that contributes to structure and large scale movement like muscle contractions

Question 24

Actin monomer

Answer 24

-aka monomeric actin protein
-basic building block of the actin cytoskeleton
-cells can express different types which allow cell to match the monomers to specific functional needs
- barbed (+) end and pointed (-) end

Question 25

Actin filaments

Answer 25

-actin monomers come together to form long thin, actin filaments in short
-actin monomers are binding to each other longitudinally and laterally (similar to double coil structure)
-combination of longitudinal and lateral bonding -> high tensile strength (resistance to breaking under tension) and can withstand pulling forces that would pull microtubules apart

-polarized
-note end names barbed (+) and pointed (-) derived from electron microscopic examination of shapes
-barbed apperance from assosication with other proteins like myosin

Question 26

Actin polymerization

Answer 26

-like microtubules, bind to nucleotide phosphates
-but bind to ATP/ADP
-binding ATP to actin monomer promotes assembly of polymerization
-binding of ADP discourages polymerization and may lead to the disassembly of filaments
-preference for ATP so if constant source of ATP then ADP’s are replaced by them

Stage 1: nucelation
-two actin monomers can dimerize, but nucelation occurs when third monomer binds -> nucelus trimer
-forms core on which rest filament forms
-Although structurally simplier than MTOC, serve same purpose

Stage 2: Elongation
-additional actin monomers added to nucelus
-can occur from both ends of filament starting at nucleus, so both ends can lengthen yet like polymerization, plus end favored
-also dynamic, both being added or removed
-if balance favors adding monomers -> filament elongates

Stage 3: steady state
-rate of assembly equals rate of disassembly and net actin filament elongation ceases

Question 27

Actin treadmilling and treadmilling regulation

Answer 27

-as minus end exposed, dynamic adding and removing of monomers can occur and this allows for actin filaments to undergo treadmilling
-favored additon of monomers to one end with same rate of monomer removal at other end
-allows keep at same length but result in filament moving within cell

regulation:
-by ATP-actin concentration compared to ADP-bound actin
-critical concentration of ATP-actin to polymerize is lower at plus end meaning if ATP-actin concentration just right, treadmilling
-ATP-actin concentration increases above critical point for minus end -> actin monomers can be again added to that end
-treadmilling gives ability to rapidly adjust actin cytoskeleton faster than indermediates
-monomers are adding to the plus end and leaving from the minus end of actin filaments

Question 28

Different actin-binding proteins

Answer 28

-regulate actin cytoskeleton for disassembly
-actin filaments greatly dynamic
-assembly/diassembly not random or out of control
-can be modified by phsophorlyation, alkylations, and disulphide bonds
-plus other proteins can modulate structure and functions

Monomer-binding proteins
-bind directly to actin monomers and influence polymerization

nucleating proteins
-bind to actin polymers to increase stability and allow for growth of new branch

capping proteins
-bind to plus or minus end and can stabilize the polymer to prevent disassembly or future assembly

severing and depolymerization proteins
-bind to actin polymer and sever or induce disassembly

cross-linking proteins
-allow side to side linkage of actin polymers to form bundles of actin filaments

membrane anchors
-link actin filaments to nonactin structural proteins, similar to those integral to the plasma mebrane such as integrins

actin-binding motor proteins
-bind to actin filaments and allow movement

most common: myosins
-18 families of myosins, each with specific role in speciic cell type
-multi-subunit proteins
-different subunits either light chains or heavy chains based on relative size
-organized into 3 domains
1. Motor- motor domained, formed by heavy chain, binds to active filament and ATP
2.Regulatory- formed by heavy chain and two light chains, moves back and forth as myosin moves along filament
3.tail-tail domain binds to other cellular proteins or other myosins

myosin movement is a energy-dependent process requiring ATP

hydrolysis
-ATP bound to motor domain
-myosin unbound to actin filament
-hydrolysis of ATP to ADP and inorganic phosphate causing conformational shift in regulatory domain, swinging like lever

actin binds
-motor domain binds to actin filament
-inorganic phsophate released from myosin causing another conformational change and pulling the myosin along actin filament
-ADP released
-binding of new ATP
-causes myosin to unbind from actin filament

Movement
-most instances, myosin moves towards barbed end

Question 29

Similarities between cytoskeletal components

Answer 29

intermediate filaments and microtubules and actin
-have tissue and cell-specific expression
-polymers

microtubules and actins
-polar, with plus and minus end
-associate with motor proteins

Question 30

Intermediate filaments summary (Function, Structural Units, Assembly, Binding Interactions, Stability)

Answer 30

Function:
-like bones, strong
-resistant to change in shape

Structural Units:
-monomers made of a-helices

Assembly:
-monomers, dimers, tetramers
-unit-length, immature and mature

Binding Interactions:
-keratins
-desmins
-lamins
-nestin
-neurofilaments

Stability:
-phsophorlyation
-glycosylation

Question 31

Microtubules summary (Function, Structural Units, Assembly, Binding Interactions, Stability)

Answer 31

Function:
-create cargo travel routes (cell trafficking)
-exert force for movement within cell

Structural Units:
-a-tubulin
-beta-tubulin

Assembly:
-polymer joining tube structure, assembly and dissassembly
-Binding GTP/GDP

Binding Interactions:
-MAPs, including motor proteins dynein and kinesin (bind ATP/ADP)

Stability:
-dynamic instability: catastrophe, capping, and rescue

Question 32

Actin summary (Function, Structural Units, Assembly, Binding Interactions, Stability)

Answer 32

Function:
-large scale, whole cell movements (ex. muscle contraction)

Structural Units:
-monomers
-filaments with longitudinal and lateral bonding- high tensile strength in a double coil

Assembly:
-polymerizes in three stages: nucelation, elongation, and steady state
-barbed and pointed ends
-bind ATP/ADP

Binding interactions:
-monomer-binding proteins
-nucelating proteins
-capping proteins
-severing and depolymerixation proteins
-cross-linking proteins
-motor proteins called myosins (bind ATP/ADP)
-treadmilling

Question 33

Where does most cell movement occur

Answer 33

-in cell
-almost all cells participating in some sort of movement
-big examples include muscle contractions and molecular motors

Question 34

Cellular Migration

Answer 34

Coordination within cytoskeleton
-arise from inside cell as all forces that underlie migration must originate from cell itself

rapid and dynamic assembly and disassembly
-ex. assemble to filaments and back into protein monomers or dimers quickly
-essential to generate forces and coordination necessary for cell migration

Question 35

What does a cell use to move? and begin?

Answer 35

-actin filaments and associated motor protein to generate initial pushing and pulling forces
-initiated when actin polymerize near plasma membrane and physically push it outwards
-pushing force of extending actin filament on plasma membrane not rip membrane due ot hydrpohobic interactions between membrane phospholipids ho,ding it together

Question 36

Types of actin filaments in cellular migration

Answer 36

-3 types which can push against membrane
-cytoskeletal remodelling may need to occur throughout cytosol during movement and not just at the leading following edges of cell

Filopedia
-thin, parallel bundles of actin filaments
-all have same polarity with plus ends facing membreane
-extend in direction of intended motion
-very end of “feet”

Lamellipodia
-larger, sheet-like bunches of actin filaments
-polar with plus end towards membrane
-form broader structures that distend a wider amount of plasma membrane in same direction as filopedis
-located above filopedia almost more in middle
-as filopeida and lamellipodia extend, plasma membreane integrins bind to extracellular matrix
-internally, actin filaments bind to integrins as anchors

stress fibres
-form around integrins
-resemble filopeida but different polarity, plus ends oriented towards cytosol
-rich in motor proteins
-anchored to integrins that allow actin filaments to move forward
-at trailing edge of cell, integrins internalized and recylced and actin stress filaments dissembles so actin monomers may be used elsewhere.

Question 37

Define cell cycle

Answer 37

-series of phases which all cells pass through in order to divide
-describes activities of cells at every stage in their lives
- two overachieving phases: interphase and mitosis

Question 38

interphase

Answer 38

-made of up 3 phases: G1, S, & G2
-most of cells like occur in G1
-not dividing cells, either actively living or preparing to divide

Question 39

mitosis

Answer 39

-cells dividing which requires lots of energy
-stimulus of signal -> cell starts dividing and go through cell process clockwise
-process continously repeated through cells life

Question 40

purpose of cell cycle checkpoints

Answer 40

-during division, cell processes stop, massive rearrangement occurs, risky
-control points of transitions between phases to avoid unnecessary energy waste
-4
-functional unit checkpoints: cyclins and cyclin-dependent kinases (CDKs)
-cyclins a class of protein associated with progression through cell
-cyclin binds to respective cyclins to become activated
-once activated, kinases phosphorylate other protein and trigger next step in cell cycle

Question 41

cell cycle

Answer 41

ALL OF THESE STEPS EXCEPT INTERPHASE EXCEPT MITOSIS
G1 Phase: Gap 1
-cells active and growing but not committed yet to undergo division

G0 Phase: Gap 0
-technically not part of cycle
-occurs when cells not dividing
-“resting”
common ex. muscle and nerve cells

G1/S Checkpoint
-cell proteins checked for DNA damage
-known as start point
-commits cell to progression throughout cycle
-activates range of signals that allow cells to divide
-p53 occur in

S Phase: Synthesis
-cell replicates entire genome in preparation for division
-centrosome of cells duplicated

S/G2 Checkpoint
-DNA integrity checked

G2 Phase: Gap 2
-last chance for cells to grow before dividing
-total amount of cytoplasm and cellular contents like endomembrane system increased in preparation for division

G2/M Checkpoint
-triggers large scale rearrangement to structure of cell, which facilitates mitosis
-increase in cell volume causes progression through this checkpoint

M phase: Mitosis
-stage where mitosis (division) occurs
-parent cells divide into 2 daughter cells
-many drastic changes like protein syntesis haulted, endomembrane system, cytiskeleton, and cell membrane all reorganzied and nucleus dissolves
-risky phase as error in any step result in defective cells

Mitotic Spindle Checkpoint
-ensure all chromosomes properly separated
-prevents chromosome imbalance
-ensures cytokinesis (division of cytoplasm) only occurs after mitosis is successfully completed

Question 42

P53 Protein

Answer 42

-a tumour suppressor protein that ensures cells with damaged DNA don’t divide
-initiate apoptosis in cells with damaged DNA
-if this protein is dysfunctional -> cells aquire ability to evade apoptosis and replicate uncontrollably
-over 50% of tumours have a mutation in the gene that codes for p53
-li-fraumeni syndrome is if there is a mutation in that protein

Question 43

what do cancer cells have the power to do

Answer 43

-evade apoptosis and manipulate cell cycle to facilitate continuous replication

Question 44

Mitosis steps

Answer 44

Prophase
-first step

chromosome condensation
-chromosomes densely condensed and packaged into chromatids
-each chromosome replicated so 2 copies called sister chromatids
-connected at structural point called centromere (region which hold sister chromatids together)
-centromere also where kinetochore (located near centromere, location of attachment of spindles during cell divison) and mitotic spindle attaches during cell division
-gene transcription shut down, entire endomembrane system dissolves into tiny vesicles
-only mitochondria remain intact
-vesicles and mitochondria randomly distributed throughout cell, so each daughter cell have organisms necessary for life

after condensation
-nucelar envelope dissovlves, releasing chromosomes into cytosol
-microtubule network of cell reorganies to form mitotic spindle, which forms around each of two centrosomes
-centrosome duplicated in S phase
-two structures- centrioles and additional proteins form centrosomes- which are MTOCs
-centrosomes not to be confused with centromeres on chromatids (region which holds sister chromatids together)
-centrosomes (responsible for microtubule organization in mitosis that arranged microtubules consisting of two centrioles) moved to opposite ends of cell by tubulin and motor proteins

Prometaphase
-kinetochore forms which a protein complex that binds to chromatids on either side of centromere
-each pair of sister chromatids will have two kinetochores, one of either side
-kinetochores function as molecular motors
-use ATP to polymerize and deploymerize the microtubule spindle fibres, allowing chromosome to move in cell
-chromosomes move to center of cell

Metaphase
-chromosomes at spindle equator, equidistant to two centrosome poles (look like laying down)
-chromosomes attached to kinetochore microtubules, which pull equally in both directions
-cell checkpoint mitotic spindle checkpoint
-chromosomes must be properly aligned at spindle equator can cell enter next stage

Anaphase
-proteins that bind sisters cleaved, dividing chromosomes into 2 identical daughters
-kinetochore microtubules shortened, movin chromosomes apart and towards opposite ends of cell
-in late anaphse, chromosomes reach max condensation level in prep for final stage of cell division
-nucleus reforms
-additonal microtubules begin to organize around spindle equator, in prep of development of contractile ring of cytokinesis

Telophase
-final step is reorganization of cell
-rearrangements that occured in prophase reversed
-nuclear membrane reformed around chromosomes
-interphase cytoskeleton begins to reform
-endomembrane system begins to reform

cytokinesis
-final step in cell division but not step in mitosis
-in mammalian cells, contractile ring forms wjere spindle equator used to be located
-as ring tightens, cel divided roughly in half
-as close, brief moment when plasma membrane snaps
-however, hysrophobic nature of plasma membrane cause rupture to reseal spontaneously
-once occured, 2 new cells enter interphase to reform cell junctions and the interior architecture of normal cell