Cell Bio Chapter 17

Motor proteins 1. Motor proteins bind filaments (microtubules, actin microfilaments), and they burn energy in order to travel up-and-down them in carrying cargo with them as they go. How do they "get" this energy? 2. Differences in motor proteins are based on what 3 things?

1. ATP hydrolysis 2. the type of filament they bind, the direction in which they travel, and the cargo they carry.

Actin Microfilaments Cell motility is a ____1___ regulated process. As calcium levels rise, the ______2______ binds to calcium which then can bind to ____3____, which ______4_______ the myosin light chain. Result: it allows _____5______ to interact with actin microfilaments. If it doesn't get phosphorylated, then it will not interact with the actin microfilaments. A counter-balancing myosin light chain phosphatase keeps the light chain _______6________ under normal conditions (high/or low Ca2+ concentrations? ((7)).

1. Ca2+ 2. calmodulin 3. MLCK 4. phosphorylates 5. myosin 6. dephosphorylated 7. low Ca2+ concentrations

What are the 4 main functions of Microtubules?

1. Generation of polarized cell types 2. Vesicle transport 3. Ciliary and Flagellar Movement 4. Mitotic Spindle/Mitosis

What are the 4 functions of Actin Microfilaments?

1. Generation of the Cell Cortex 2. Cell Motility 3. Muscle Contraction 4. Cytokinesis

Intermediate Filaments There are >50 different human keratin genes, each encoding one of two protein types (1. list them) and each obeying tissue-specific _____2______ rules with other keratins.

1. I-acidic, II-basic 2. dimerization

What are the following cytoskeletal components made out of? 1. Intermediate Filaments 2. Microtubules 3. Actin Filaments

1. Intermediate filaments: fibrous intermediate filament proteins 2. Microtubules: tubulin proteins 3. Actin Filaments: actin proteins

Microtubules What are the 2 motor proteins? What filaments are each associated? What are the motor polarities of each(what direction is it moving)? What cargo does each carry?

1. Kinesin, microtubules, plus end, cargo: vesicles, organelles, and other microtubules 2. Dynein, microtubules, minus end, cargo: organelles, cytoplasmic (vesicles), and axonemal (cilia, flagella)

Actin Microfilaments - Motor Proteins 1. Motor proteins not only bind filaments (actin, microtubules), but they burn energy in order to travel up-and-down them in various directions carrying cargo with them as they go. How do they harness this energy? 2. Differences in motor proteins are based on what three things?

1. Motor proteins couple ATP hydrolysis with changes in conformation such that they lose contact with one subunit of the protofilament and then regain it with the next. 2. The type of filament they bind, the direction in which they travel, and the cargo they carry.

1. Do intermediate filaments have motor proteins? 2. What are the motor proteins for microtubules and actin filaments?

1. No because motor proteins require polarization which IFs do not have because they are not polar. 2. Microtubules: Kinesins and dyneins Actin filaments: Myosin

What are the three parts of the intermediate filament? provide 1-2 characteristics of each.

1. The central/rod region : a-helical and amphipathic. Drives IF self-assembly, forming coiled-coils with other IF proteins as part of the dimerization process. 2. Linkers: sub-divide the central/rod region & may be important for defining which IF proteins dimerize with each other 3. Head and tail region: they are different in length and sequence based on the type of IF that it is. sites where IFs are phosphorylated / intact with each other

1. If you have a lot of profilin in a cell, will you have a lot of actin filaments? 2. What if you have a lot of cofilin?

1. Yes you will have a lot of actin filaments because profilin builds MFs 2. If you have a lot of cofilin, the actin filaments will be broken down to G-actin monomers

Microtubules the MTs that form cell motility structures like cilia and flagella, depend on specialized _____1_____ called ______ 2_______ for MT nucleation.

1. centrioles 2. basal bodies

New microtubules radiate out from the _____1_____ with their minus ends at the _____2_____ and their plus ends extending into the ____3_____

1. centrosome 2. centrosome 3. cytoplasm

Actin Microfilaments The extracellular matrix is to the outside of the cell as the _______1________ is to the inside, ____2____ proteins that provide structural support, in this case for whole tissues. Integrin receptors and adaptor proteins (including tyrosine kinases) comprise the focal adhesions that bridge what together?

1. cytoskeleton 2. fibrous focal adhesions at the leading edge of the cell bridge actin cytoskeleton with the extracellular matrix.

Actin Microfilaments Cell motility is also regulated by Rho-type GTPases Each GTP-bound Rho-type GTPase, when activated, has a unique effect on the actin cytoskeleton Cdc42-GTP activates _____1_____ leading to ______2______ formation. Cdc42-GTP also turns on Rac GEF. Rac-GTP activates the Arp2/3 complex leading to ________3________ formation, along with a Rho GEF. Rho-GTP activates Rho kinase that independently phosphorylates the regulatory light chain of myosin stimulating cell contractility in the same way that ____6___does.

1. formins 2. filopodia 3. lamellipodia (branched at leading edge) 5. Rho kinase 6. MLCK

CYTOPLASMIC - Intermediate Filaments 1. What type of intermediate filaments are typically found in epithelial cells? 2. What type of intermediate filaments are typically found in connective tissue cells, muscle cells, and glial cells? 3. What type of intermediate filaments are found in nerve cells? NUCLEAR 4. What type of intermediate filaments are found in all animal cells?

1. keratin filaments 2. vimentin and vimentin-related filaments 3. neurofilaments 4. nuclear lamins (in nuclear envelope not cytoplasm)

Microtubules The ER is carried by _____1_____ towards the periphery of the cell while the Golgi is carried by ____2___ toward the centrosome. this creates the positioning of these organelles in the cytoplasm. periphery +/-? centrosome +/-?

1. kinesins --> periphery (+ end) 2. dyneins --> centrosome (- end)

Microtubules: Dynein 1. In isolated doublet microtubules: dynein produces...? 2. In normal flagellum: dynein causes...?

1. microtubule sliding 2. microtubule bending

The ATP-binding sight of G-actin opens on one side of the protein, the ____1____ end oppose the N- and C- termini of the protein which is the ___2____ end

1. minus end 2. plus end

Microtubules MTs are organized within the centrosome. The centrosome is comprised of many ring-shaped structures containing the protein γ-tubulin. These γ-tubulin ring complexes can each serve as a '____1_____ ________' for the formation of new MTs. MT extension (growth) is almost exclusively at the __2__ end of each protofilament. MTs thus exhibit ___3____ growth.

1. nucleation point 2. plus 3. polarized

Intermediate Filaments Describe the formation of an IF (2 steps)

1. one monomer forms coiled coil between other monomer (head to head, tail to tail) 2. one dimer links on to another dimer laterally to form a staggered tetramer --> (head to tail, head to tail) which causes the IF to have no polarity.

Intermediate Filaments IF proteins dimerize into coiled coils in _____1_____ fashion, thus maintaining the _____2_____ of individual monomers

1. parallel (head to head and tail to tail) 2. polarity

Actin Microfilaments Most of the monomers that decide to jump onto a filament jump onto the ____1___ end. When it adds onto the plus end and becomes a part of the protofilament, its ATPase activity increases which allows it to....? (2) The G-actin monomers bind very quickly so that they are still bound to ATP and have not hydrolyzed it yet. This is called an _____3_____ and it is a sign that filament is ____4____. it is analogous to....? (5) Eventually as you go to the ___6___ end to the ___7___ end you will find an individual subunit that has finally hydrolyzed its ATP to ADP. Individual subunits do not immediately release the energy provided by ATP hydrolysis; rather it is stored within the subunit as bound ADP and inorganic phosphate.

1. plus end 2. hydrolyze its ATP to ADP. 3. ATP-cap 4. stable 5. GTP-cap (microtubules) 6. plus 7. minus

Intermediate Filaments What are the 3 purposes of Lamins?

1. protection and stabilization of the nucleus 2. bind to and regulate the expression of genes (close to the DNA) 3. Help DNA be repaired from damage

G-actin monomers polymerize in the same direction such that the resulting ______1________ retains the same ____2____ of the monomer. 3. Two, parallel actin protofilaments wrap around each other in a right-handed helix to form what?

1. protofilament 2. polarity 3. F-actin

Actin Microfilaments (MFs) (F-actin) F-actin is comprised of two ________1________ that wrap around each other in a helical pattern. ALL F-actin protofilaments have the same orientation so F-actin is _______2______ with minus and plus ends. G-actin generally associates with protofilaments at the ___3___ end and dissociates at the ___4___ end. G-actin is an ____5_____ B-tubuliun is an _____6_____

1. protofilaments 2. polarized 3. associates - plus end 4. dissociates - negative 5. ATPase 6. GTPase

Actin Microfilaments - Cell Motility Reach: Reaching forward and forming ____1_____ (filopodia, lamellipodiais) an actin ____2____ process at the LEADING edge of the cell Grab: After reaching out, ________ 3___________ enable cells to grab onto the underlying ______________ 4__________ Squiggle: Cell squiggling depends on the contraction of actin microfilaments, driven by the motor protein ___5____.

1. protrusions 2. actin treadmilling process 3. focal adhesions 4. extracellular matrix 5. myosin

Actin Microfilaments In the absence of nucleotides, myosin locks tightly to actin, a state called ____1____; it only lasts for a short part of the cycle due to....? ATP binds weakly to myosin at a large cleft opposing the actin-binding site, leading to slight changes in the conformation of the "_______3 ________" and detachment of myosin from _____4_____. Gross changes in conformation follow as the cleft closes around the bound ATP and the "neck region" swings forward at an angle of 70 degrees and across a distance of 5nm. ATP hydrolysis follows, but both ADP and Pi remain bound. Weak binding of myosin to actin occurs one position further up on the microfilament towards the plus end. Weak binding to actin triggers release of ___5___. This triggers _____6_____ binding by myosin to actin and a so-called power stroke where the lever arm swings back to its original position and ____7____ is released. A state of rigor is regenerated one position closer to the ____8____ end of the microfilament.

1. rigor 2. high cytosolic levels of ATP 3. "head-region" 4. actin 5. Pi 6. tighter 7. ADP 8. plus

Actin Microfilaments - Muscle Contraction The ______1______ is an actinomyosin superstructure that coordinates cell contractility across the entire tissue. Ca2+ activates the Ca2+-binding proteins tropomyosin and troponin C (part of the troponin complex). Both of these bind to calcium, when they are not bound to calcium, they are tightly bound to what? (3) Calcium regulates this process by releasing the actin microfilaments from the proteins that tend to smother it.

1. sarcomere 2. Tropomyosin 3. actin microfilaments

Actin Microfilaments - Generation of Cell Cortex Actin accessory proteins like ____1____ help cross-link actin microfilaments to transmembrane proteins creating a cell cortex This is analogous to the intermediate filament proteins of the ______ _______

1. spectrin 2. nuclear lamina Actin Microfilaments - cell cortex Intermediate filaments - nuclear lamina

The dynamic rearrangement of actin microfilaments is called ______1_______ This process involves the simultaneous ____2____ of monomers at the plus end and ___3___ of subunits from the minus end such that the net length of the MF may not change

1. tread milling 2. gain (+ end) 3. loss (- end)

1. Microtubules are POLYMERS composed of...? 2. Each tubulin subunit is a heterodimer comprised of the globular proteins...? 3. Each tubulin subunit associates longitudinally with other tubulin subunits in the same orientation such that the entire "_______________" is polarized with an a end and a B end. What are the charges for each of the ends? 4. These protofilaments then align ____________ with other protofilaments (13 in total) to form a hollow cylinder of 25 nm diameter

1. tubilin particles (protein tubulin) 2. a-tubulin and B-tubunlin 3. protofilament a(minus) end// b(plus) end 4. laterally

Referring back to the original figure again, which letter designates the direction in which myosin II would direct actin microfilament movement keeping in mind that myosin is fixed to the slide? A. A B. B C. C D. D (slide 85/85 Ch. 17)

A. the minus end

The cytoskeleton comes in three flavors: list them

Actin Microfilaments (MFs) Microtubules (MTs) Intermediate Filaments (IFs)

Actin Microfilaments protein complex that structurally mimics G-actin structure and nucleates new MF formation from existing MFs; also caps minus ends

Arp2/3

Which of the following best depicts what would have happened if you had also added an excess of plus end capping protein at the same time in which the actin monomers and ATP were added? A. A B. B C. C D. D E. E (slide 84/85 of Ch. 17 ppt)

C. capping proteins tend to bind bind to the ends of actin microfilaments and block whatever is trying to be bound Actin microfilaments PREFER to add to the positive end but it CAN add to the negative end. SO if you add a capping protein on the plus end it is FORCED to add onto the minus end, which may not make it as stable but it is possible

You conduct the following experiment. First, you link a short actin microfilament to myosin II, which had been pre-bound to a slide. You then add an excess of actin monomers and ATP to the slide and allow actin polymerization to proceed for a few minutes, after which an image of the structure is taken by electron microscopy. Which best designates the plus end of the actin microfilament? A. A B. B C. C D. D (picture on slide 83/85 of Ch. 17)

C. When you add actin ATP, you see the assembly going toward to C. Assembly starts at the plus side so side C is where the plus end would be located.

Cytoskeletal filaments that display 'dynamic instability' __________. A. constantly slide past each other. B. move around in the cytoplasm with a whip-like motion. C. change in length due to polymerization and depolymerization. D. tend to make the cytoplasm dynamically unstable.

C. change in length due to polymerization and depolymerization. (microtubules)

Actin Microfilament twists and destabilizes MFs; binds minus end ADP/Pi-bound subunits to speed dissociation

Cofilin

Microtubules Colchicine and taxol are two drugs that inhibit mitosis by interfering with the development of the mitotic spindle. Each of these drugs binds tightly to existing microtubules causing what to happen...? (opposite effects)

Colchicine: causes the microtubules to shrink but not grow Taxol: causes the microtubules to grow but not shrink

What would lead to microtubule stability? A. Application of the non-hydrolyzable GTPγS to cells. B. Application of the drug taxol to cells. C. Application of an siRNA to catastrophin. D. All of the above.

D. All of the above.

Which of the following statement correctly describes intermediate filaments? A. They are the only cytoskeletal filaments that are not composed of proteins. B. They are the principle components of the cell cortex. C. They are always found outside of cells. D. They help the cell to withstand mechanical stress.

D. They help the cell to withstand mechanical stress.

Intermediate Filaments Keratins help link individual epithelial cells together with similar cells in epithelial tissues like the skin. _________________ serve as anchorage points for these cells.

Desmosomes

Linear arranged MFs are formed through formin-mediated MF nucleation

Filopodia

Actin Microfilaments nucleates new MFs, but in a linear fashion

Formins

Microtubules All tubulin proteins bind GTP, and β-tubulin is capable of carrying out GTP hydrolysis. Switching between GDP and GTP bound states has important implications for MT growth and stability. GTP-bound β-tubulin promotes what? GDP-bound β-tubulin promotes what?

GTP-bound β-tubulin promotes microtubule linearity and growth (GTP cap) GDP-bound β-tubulin promotes microtubule curvature and shrinkage (GDP tubulin)

Using radiolabeled cholchicine as a probe, who isolated these particles, called tubulin, from purified spindles, sea urchin eggs, cells in tissue culture, and brain tissue (1967)?He later showed, as suggested by Inoue, that microtubules can be made from individual tubulin subunits.

Gary Borisy

Actin Microfilaments The drugs Iatrunculin and phalloidin both inhibit cell motility by interfering with the rearrangement of actin microfilaments. What do each do? (opposite effects)

Iatrunculin - binds tightly to actin monomers preventing their polymerization (sea sponge) Phalloidin - binds tightly to F-actin and stabilizes it against depolymerization (locks cells and keeps your cells from moving and dividing)

Which are these are polar? Which are not polar? Intermediate filaments microtubules actin filaments

Intermediate filaments - not polar Microtubules - polar Actin Filaments - polar

Intermediate Filaments ____________ are found in every epithelial cell of the body. They are the most diverse IF protein family

Keratins

Microtubules Kinesins are ______ end motor proteins Dyneins are ______ end motor proteins Which is faster? What are the two classes of Dyneins?

Kinesins are plus end motor proteins Dyneins are minus end motor proteins Dyneins are faster because they contain ATPases 2 classes 1. Cytoplasmic - intracellular transport, cell polarization, and cell motility 2. Axonemal - ciliary and flagellar beating

Branched actin microfilaments that form at the leading edge through Arp2/3 complex driven nucleation

Lamellipodia

Intermediate Filaments What IF has a function in the protection and stabilization of the nucleus?

Lamins

Intermediate Filaments What is the only nuclear localized IF protein? These line the inside of the nuclear envelope in every cell type as part of the ________ _________

Lamins nuclear envelope

Microtubules In differentiated cell types like the neuron and the epithelial cell, the MTs are oriented in such a way as to generate the unique structural elements of those cells (axons, cilia). Strategically localized _______ help ensure MT stabilization in the wake of pending ______________ in the other MTs of the cell

MAPs catastrophes

What is one thing about microtubules (MTs) that is different from intermediate filaments (IFs)

Microtubules have polarity unlike intermediate filaments

Actin Microfilaments Motor proteins? What are the motor polarity (what direction is it moving)? What cargo does it carry?

Myosin: plus end. cargo: vesicles, plasma membrane, and other actin microfilaments

Actin Microfilaments binds to ATP-bound G-actin monomers and faciliates their incorporation into existing MFs at the plus end

Profilin

Who showed that the drug colchicine leads to rapid spindle disassembly (and is accompanied by chromosome pulling towards the poles). This disassembly could be reversed without any new protein synthesis, suggesting a pool of "particles" were available for spindle reformation.

Shinya Inoue

How do intermediate filaments get their name?

They are intermediate in diameter (10nm) to the larger microtubules (25 nm) and the smaller actin microfilaments (7 nm)

T/F the cytoskeleton is neither rigid nor static

True

T/F All individual intermediate filaments are long and fibrous, largely a-helical, and dimerize with other IF proteins

True bihh!!!! we gettin' an A+ biihhhhhh okurrrt

Who was responsible for the discovery of the spindle apparatus, the reorganized version of microtubules as they exist during mitosis?

Walther Flemming

Actin is highly regulated by _______ _______ that bind to it

accessory proteins

From embryonic development to wound healing to immunity, cells move. It is an ________ __________ driven process.

actin microfilament (MF)

Lamin mutations, called laminopathies can sometimes encourage the premature __________ of cells and tissues. An example of this is progeroid syndromes that have been linked to mutations in Lamin A

aging

Intermediate Filaments The coiled coil formation of IF dimers is mediated by what?

amphipathic a-helices

Intermediate Filaments Lamins are disassembled and reassembled with each consecutive what? The breakdown of the nuclear lamina, which released the DNA, is regulated by what?

cell division phosphorylation

The cytoskeletal components are defined by their ______

diameter

Microtubules The growth and rapid disassembly of MTs in living cells is called what? What is the purpose of this? What is it modified by?

dynamic instability The purpose of dynamic instability is rapid cell remodeling dynamic instability is modified by capping proteins that either stabilize (MAPs) or destabilize MT polymers (catastrophins)

Of all the cytoskeletal proteins, which are are the strongest and why?

intermediate filaments are the strongest because their high tensile strength stabilizes the structure of individual cells as well as group of cells within tissues

Intermediate Filaments Lamins exist as two types? What are they?

lamin A (soluble) lamin B (membrane bound, via isoprenylation)

Where do microtubules lose their length? Where are they stabilized? This opposes actin filaments

lose their length: + end stabilized: - end

Intermediate Filaments IF proteins are prominent in the cells of tissues subject to what? Give 3 examples

mechanical stress skin - epithelial cells - keratins muscle - muscle cells - vimentin/desmin nerve - neuron - neurofilaments

Intermediate Filaments A mutation of Keratin 14 causes what?

mild blistering

Dynamic rearrangements enable gross changes in morphology necessary for basic cell processes from ________ to ________.

mitosis to motility

Microtubules Vesicles and some organelles move in directed fashion in cells along MTs, which form an internal highway system of sorts for intracellular transport. The drivers of such transport are a couple of types of specialized ATPases called...? backward transport of minus end motor proteins goes to the..? outward transport of plus end motor proteins goes to the..?

motor proteins (kinesin and dynein). backward = cell body outward = axon terminal

Intermediate Filaments Things that are polar will/will not go through a membrane things that are non polar will/will not go through a membrane

polar: will not nonpolar: will

Accessory proteins control what three things in actin microfilaments?

synthesis breakdown three-dimensional organization of MFs in the cell.

Actin microfilaments (MFs), or filamentous actin (F-actin) are polymers whose minimal subunit is...?

the globular protein G-actin.

Intermediate Filaments What causes IFs to have no polarity?

their anti-parallel orientation

Actin Microfilaments 1. Myosin are locked in position but they have motor ability. which means that as it moves to the plus in the actin microfilament slides 2. Myosin bind to parallel actin microfilaments that are in opposite orientations. and as they start to "walk" they slide the actin microfilaments toward each other.

ya