Lecture 4

Question 1

What microscope techniques can you NOT use to see cytoskeletons?

Answer 1

Light microscope cannot be used, it’s diameters range from 7nm to 25nm, and the resolution limit is about 200 nm and it limits the wavelength of visible light.

Question 2

What microscope techniques can you use to see cytoskeleton?

Answer 2

1) Flurocent microscopy- it uses the same resolution as light microscopy however we can fluorescent label to detect specific proteins.

2) Transmission electron microscopy: uses beams of electron, very short wavelength and the resolution limit is about 1nm. It reveals detailed structure

Question 3

What is immunofluorescene microscopy used for?

Answer 3

It is used to determine location of proteins within the cell

Question 4

How does Immunofluroescenence microscopy work?

Answer 4

It is used to determine the location of a specific protein, and the cells are fixed (they are not imaging)

Primary antibody is used to bind to specific protein of interest and then secondary anti body binds to the primary antibody. (This is covalently tagged to the flurocent marker)

Then fluorescence microscope is used to excite fluorescent marker and visualize the light emitted.

Question 5

What are the 3 types of filaments that form the cytoskeleton?

Answer 5

The smallest ones are Actin which are made of actin filaments

Next is Intermediate filaments which are made up of intermediate filament proteins and the largest are micro tumbles which are made up of tubulin

Question 6

What sort of interactions are the microtubles filaments held together by?

Answer 6

Non covalent interactions

Question 7

What are the four kinds of intermediate filaments

Answer 7

Cytoplasmic:

Keratin filaments (found in epithelial cells)

Vimentin and vimentin-related filaments ( found in connective tissue cells, muscles cells, and glial cells)

Neuro filaments (in nerve cells)

Nuclear:

Nuclear lamins (found in all animal cells)

Question 8

What are the two types of intermediate filaments and what are their functions?

Answer 8

These are involved in structural support. And the cytoplasmic IFs: are in animal cells subjected to mechanical stress and they provide mechanical strength

Nuclear IF: they are in nuclear lamina and provide 2D mesh work. Formed by lamins in all animal cells (plants have different lamin-like proteins)

Question 9

What are the structural characteristics of cytoplasmic intermediate filaments

Answer 9

These are conserved alpha helical central rod domain and has a different C and N terminals.

However, a monomer with polarity (different ends ) coils with another monomer making a dimer which is still polar. These are staggered anti parallel tetramer.

Then 8 tetramers associate side by side and assemble into filaments. And the inter actions are mostly non covalent, and there is no filament polarity.

Now this structure is tough, flexible and high tensile strength.

Question 10

Give a specific keratin filament name in the epithelial cells and where they are found

Answer 10

keratin filaments in epithelial cells form a network throughout cytoplasm out of cell periphery.

They are anchored in each cell at cell-cell junctions and connect to neighbouring cells. They provide mechanical strength

Question 11

What are microtubles involved in?

Answer 11

These are in all organizing functions is all eukaryotes. They are involved in cell organization like vesicle transport, organelle transport and positioning, and animal cells near centromers

They are involved in mitosis and structural support ln cells and motile structures (flagella, cilia)

These are made up of tubulin so they are long and stiff, hence INEXTENSIBLE

Question 12

How is the structure of the microtubles made?

Answer 12

These are long hallow tubes that are made of subunits of individually two close related globular proteins which are alpha tubulin and beta tubulin. They both first bind to GTP and then bind to each other to form a tubulin hetero dimer.

This regular arrangement gives alpha and beta subunits the polarity, and 13 parallel prototilaments make up a hallow tube

Question 13

What is the plus end and the minus end in microtubles?

Answer 13

Beta end is the plus end and minus end is the alpha end

Question 14

What type of bonds are between the individual subunits of microtubles?

Answer 14

Non covalent

Question 15

Which bonds are weaker in microtubles?

Answer 15

Between protofilaments are weaker than the bonds within each protofilaments

Question 16

Which side is grow to faster in microtubles

Answer 16

It occurs both sides but is faster on the plus end

Question 17

When is a microtubule called D-form dimer?

Answer 17

When the protofilaments has been in the tubulin for a while beta tubulin will cut GTP to GDP and that will be called D-form Herero dimer

Question 18

Is T-form heterodimer growth or shrinkage

Answer 18

T-form is growth while D-form is shrinkage

Question 19

What is the role of microtubles organizing centres?

Answer 19

In a cell MTOCs are centrosomes in animal cells, the minus end is stabilized at the MTOC and the plus end keeps growing end.

Question 20

What is dynamic instability?

Answer 20

The plus ends of the microtubles can shrink or grow this is called dynamic instability

Question 21

When does the GTP cap occur?

Answer 21

If the hydrolysis of GTP is newly added alpha beta tubulin diners is slower than the addition of the a-b- tubulin diners, it leads to the formation of GTP-cap and stabilizes the plus end. And the microtubles continue to grow.

Question 22

Why is there disassembling in Microtubles?

Answer 22

In GDP tubulin diners; there is conformational change and weaker binding which curves the filaments and disassemble.

Question 23

What sites does MTOCs have for microtubule growth?

Answer 23

They have Nick eating sites that have gama-ring complexes and accessory proteins. They start assembling new microtubule. The minus end is with gamma-ring complexes and act as attachement sites for the a-beta-tubulin diners

Question 24

Where do microtubles come from in a non dividing animal cells? In interphase

Answer 24

Most microtubule radiate from one centrosome (MTOS)

Question 25

How many centreomeres does a dividing cell have?

Answer 25

It’s centrosomes duplicate to form two spindle piles and the microtubule are reorganized to form a bipolar nito tic spindle

Question 26

What are some roles of the microtubule associated proteins?

Answer 26

Nucleate growth of new microtubule
Promote microtubule polymerization
Promote microtubule dissemble
Stabilize microtubule and prevent disassembly

Question 27

In differentiated cell like a neuron how can cell transport its cargo?

Answer 27

Via the motor proteins on microtubule

Question 28

What are the two types of motor proteins in a cell?

Answer 28

Kinesines (kinesin 1) generally move towards the plus end of the microtubule an and transport cargo of organelles, vesicles and macromolecules.

Dyneins: move towards minus end of microtubule, like cytoplasmic dynein that moves towards minus end of the cell body and take cargo of worn out mitochondria and endocytosed material.

Question 29

How do kinease 1 and dynein dimers move in the microtubule?

Answer 29

They use ATP hydrolysis and the heads move along microtubule a abs tails transport cargo.

Question 30

What are actin filaments also called

Answer 30

Microfilaments

Question 31

What are actin or microfilaments made of?

Answer 31

Actin molecules and they are flexible however inextensible

Question 32

What are the motor tubules in actin or microfilaments

Answer 32

Myosins

Question 33

Since actin is also helical is it dimers? And what sort of interaction does it have?

Answer 33

No it is composed of single type of globular protein called actin monomer and they have non covalent interactions.

Question 34

Does actin have polarity?

Answer 34

Yes it has different ends cause all the actin monomers are in the same orientation in each protofilament and the growth is faster on the plus end

Question 35

What does actin monomer bind to in order to form a monomer?

Answer 35

It binds to ATP. Just like on the case of microtubules, if ATP hydrolysis is after then it reduces the strength of binding between monomers in filament and rapid addition of actin monomers will increase the Aagai cap

Question 36

What is treadmiling

Answer 36

It is the loss of actin monomers on the minus end and Addition of monomers on the plus end vein equal

Question 37

What are the steps in actin polymerization in a test tube

Answer 37

First you add actin subunits and ATap to the test tube then the nucleation phase begins (lag phase) when small oligomers form but are unstable,

Next elongation begins because some oligomers become more stable, leads to paid filament elongation (at the plus end) and then

Steady state that results in treadmilling

Question 38

What is cell crawling

Answer 38

It’s the dynamic changes in actin filament an example is where actin filaments undergo treadmilling

Question 39

What are the functions of actin filaments

Answer 39

Sequester actin monomers to prevent polymeization

Promote Nucleation to form filaments
Stabilize actin filaments (capping)
Organize : bundles, cross link filaments
Severe actin filaments

Question 40

Which direction does myosins move in actin? And how many types are there

Answer 40

They move towards the plus end and heads move along the actin filament, and use ATP hydrolysis for movement

myosin 1: tail domain binds to the cargo (can change the cell shape)
Myosin 2:

Dimers will assemble as a coiled coil, and make myosin-2 filaments like bio polar myosin-2 filament which slides a tin filament in opposite directions to generate contractile force. (It slides actin in different directions )