Cytoskeleton and Microtubules

What role does the cytoskeleton play in a cell?

It can help with the organization and movement of organelles and with cell shape, motility, and contraction

What are the physical properties of the cytoplasm?

• Finite volume → 1×10^-11 uL to 0.5 mL; average mammalian cell is ~ 4×10^-12 L

• Concentrated → 200-300 mg protein/mL; this concentration is different than what in vitro experiments usually work with (1 mg/mL)

• Complex, partially defined solution → around 20,000 different proteins present

• Non-homogenous solution → molecules are not evenly distributed throughout the cytoplasm, they are often localized

• Active matter → constantly changing and moving due to metabolic processes where proteins change shape and heat energy is released

• Characteristics of both gel and solution

• Viscoelastic → if you pull on cytoplasm, it will lengthen rapidly, and once it is released, it will slowly return back to its original shape

• Low microviscosity/high macroviscosity → free diffusion only for molecules <10 nm; motion of larger molecules is limited (more viscous, less fluid)

What was the evidence for a cytomatrix in cells?

1. Microscopy → put nonionic detergent on cells, which solubalized the membrane, then looked at cells under EM and saw a network of filaments; indirect immunofluorescence microscopy showed this network as well; GFP fluorescence microscopy showed this network too, but this time in living cells

2. Particle Diffusion Rates → small and large particles were observed to diffuse at different rates; figured larger particles were getting trapped in the lattice of filaments

What was the original definition of the cytoskeleton?

Whatever was left over after treatment of cells with nonionic detergent

What are the 3 filament networks of the cytoskeleton and their subunits?

• Intermediate filaments → intermediate diameter, 10 nm; many different proteins make these up

• Microtubules → largest diameter, 25 nm; subunits are tubulin

• Microfilaments → smallest diameter, 7 nm; subunits are actin

Are microfilaments only in the cytoplasm?

no, they have also been found in the nucleus of some cells

Are microtubules found in prokaryotes?

No, they are generally considered distinguishing features of eukaryotic cells, however this is still an active area of research

What is the general structure of microtubules?

They are hollow cylinders of variable length and they are assembled from alpha and beta tubulin subunits that form a protofilament, and 13 of these protofilaments come together to form a singlet microtubule

What are the main functions of microtubules?

• Chromosome segregation during mitosis and meiosis

• Transport of membrane-bound organelles

• Cell motility and morphogenesis

• Ciliary and Flagellar Beating

What is the structure of the tubulin subunits and how do they polymerize?

There are alpha and beta tubulin subunits that dimerize to form a heterodimer; There are G-proteins (not small-monomeric) attached to these subunits that can bind GTP and GDP; the alpha subunit is ALWAYS GTP bound, as the GTP plays a structural role; the beta subunit can be either GDP or GTP bound; if athe heterodimers are at a concentration higher than 10 uM, they will begin to bind to each other in an end-to-end arrangement called a protofilament; these protofilaments can have lateral noncovalent interactions with one another and 13 of these protofilaments will come together to form a hollow cylinder, which is a singlet microtubule

Can you see alpha tubulin in a cell without beta subunit?

No, the association of a-tubulin with B-tubulin is so tight that they are always in their heterodimer state

Are singlet microtubules the only kind of microtubules?

No, in cilia and flagella there are doublet microtubules that have the 13 protofilaments of a singlet microtubule, plus 10 additional protofilaments forming another hollow ring; centrioles are triplet microtubules that have 3 rings

What is the critical concentration for tubulin?

10 uM

What is critical concentration and how does it relate to polymerization of tubulin into microtubules?

Critical concentration (Cc) is the minimum concentration of tubulin dimers necessary form the dimers to begin forming into microtubules; if the concentration of tubulin is below Cc, then microtubules will not form; if the concentration of tubulin is above Cc, then microtubules will begin to form; after Cc has been reached, if more tubulin is added to the solution, the concentration of tubulin dimers in the solution will remain constant because new tubulin being added will be incorporated into microtubules instead of remaining as free dimers

What is the cause of the lag phase in the formation of microtubules (or any cytoskeletal filament)

It takes times for dimers to find each other and begin assembling into protofilaments

What is the cause of the plateau phase in the formation of microtubules?

The overall total mass of microtubules stays the same once many microtubules have been formed and this is due to dynamic instability (as one grows, another shrinks)

Do microtubules have polarity?

Yes, they have a plus and a minus end (has nothing to do with electric polarity)

What end of a microtubule does most growth occur at and why?

The plus end; the Cc of assembly is lower at the plus end than at the minus end, so growth will occur first at the plus end because less dimers are necessary for microtubule to form/grow

What end of microtubules is closest to the plasma membrane?

The plus end

How are microtubules arranged in the axon of a neuron?

All the plus ends are towards the distal end of the axon

What is dynamic instability?

Microtubule length is extremely variable and can change quickly; catastrophe is the disassembly of a microtubule; rescue is the assembly of a microtubule; the total mass of microtubules in a cell remains the same though, so as one microtubule shrinks, another one must grow

What do the rates of the removal/addition of different beta subunits onto the plus end of a microtubule look like?

• rate of GTP beta subunits added on > rate of GTP beta subunits coming off

• rate of GDP beta subunits coming off > rate of GDP beta subunits added on

• rate of GTP tubulin added on > rate of GTP hydrolysis

These 3 relationships allow for growth at the plus end; GTP-bound subunits are more likely to get added on and GDP subunits are more likely to get taken off

What does the structure of a microtubule look like at the plus end?

Most subunits near the end are GDP-bound, but a few subunits at the very tip of the microtubule are GTP-bound, allowing for growth

How does catastrophe occur?

If the pool of GTP-tubulin subunits gets depleted, before another GTP-subunit gets added on to the end of the microtubule, the GTP-subunits currently in the end of the microtubule will hydrolyze their GTP into GDP and the GTP cap will be gone and the microtubule will begin shrinking

How does rescue occur?

What are MAPs and what is their role?

Microtubule Associated Proteins; they can stabilize microtubules by preventing depolymerization; they can destabilize microtubules by promoting GTP hydrolysis and/or weakening lateral contacts between protofilaments

What are (+) TIPs ?

They are a family of MAPs, one example being EB1; they were originally thought to be associated with microtubule growth because on kymographs they were only seen during elongation phases of microtubules; (+) TIPs actually promote hydrolysis of GTP at the GTP cap of a microtubule, promoting shrinkage of the microtubule; the EB1 protein isn’t at the very end of the microtubule, rather it is a little further down on the microtubule

Do prokaryotes have a cytoskeleton?

Yes, they don’t have microtubules, but they do have cytoskeletal filaments; they have a molecule called FtsZ that structurally resembles tubulin and it forms filaments within a prokaryote; FtsZ polymers are ring shaped and help with cell division

What state are microtubules in when exhibiting dynamic instability?

they must be in steady state

What is the affect of taxol on microtubules?

It increases microtubule stability by lowering Cc

Is the FtsZ protein in eukaryotes similar to tubulin?

Structurally, yes; but in terms of sequence, no, they have very different sequences