BIOMG 1350 Lecture 6/7 - Cytoskeleton, Motor Proteins , Mutations and their Consequences

Cytoskeleton elements

Microtubules are composed to aB Tubulin dimers

• 25 nm in diameter

• Function is organization and long range transport of organelles

Microfilaments are composed of actin

• 7-9 nm in diameter

• Function is contractile machinery and network at the cell cortex

Intermediate filaments, reversibly assembled from a variety of subunits, are tensile structures that give cells and tissues their strong structural integrity. The keratins of hair and nails are the intermediate filaments cytoskeletons of dead cells

Microfilaments and microtubules are more dynamic than intermediate filaments, as they are subject to more frequent assembly and disassembly.

Microtubules

Microtubules, reversibly assembled from a polymer of a/B-tubulin, provide a framework for the internal organization of interphase cells and the spindle in mitotic cells.

The end with exposed B-subunits is called the plus end, whereas the end capped by a-subunits is called the minus end.

Alpha tubulin is negatively charged and beta tubulin is positively charged

Microtubules are polarized, with the + end generally favoring faster assembly and the − end generally being more prone to disassembly. When the − end is nucleated and anchored by γ-tubulin, it is stabilized, so microtubule assembly and disassembly occur primarily at the + endthe minus end.

Microtubules transport cargo along nerve cell axons through both inward and outward transport and outward transport occurred at a fast speed of 5 micrometers/second.

Centrosome (in relation to Microtubules)

Centrosome serves as the microtubule organizing center and they extend from this microtubule organizing center in both interphase and mitosis (playing a large role in pulling the chromosomes to opposite sides of the dividing cell)

Microtubules usually grow out of the centrosome or microtubule organizing center. The minus ends of microtubules are localized at the MTOC and the plus ends radiate out from the cell center of an interphase cells

In the centrosome, y tubulin ring complexes nucleate microtubules and stabilize the minus ends so the microtubules can grow without decaying

Microtubule Heterodimer

The a- and B- tubulin subunits come together to make a tubulin heterodimer (or a microtubule subunit) and they alternate down a protofilament. The protofilaments in a microtubule all have the same orientation, so in a microtubule the a-subunits are exposed on one end, and the B-subunits on the other.

Microtubule Protofilament

In a microtubule there are 13 protofilaments that make up the microtubule tube (13 protofilaments that make up the ring if we were to take a cross section of the microtubules.

Each of these protofillaments are made of alternating heterodimers.

Y Tubulin

A specialized member of the tubulin protein family that, unlike α- and β-tubulin, does not form the microtubule wall itself but instead serves as a nucleator for microtubule growth.

It typically functions as part of the γ-tubulin ring complex (γ-TuRC), located at microtubule-organizing centers such as the centrosome, where it templates the formation of new microtubules by providing a structural base onto which α/β-tubulin dimers can add.

By controlling where and how microtubules initiate, γ-tubulin plays a key role in organizing the cell's cytoskeleton, particularly during processes like mitotic spindle formation.

Alpha and Beta Tubulin Characteristics

Tubulins are GTP binding proteins.

• GTP bound tubulin is more stably associated with microtubules than is GDP bound tubulin. The GTP in B tubulin is slowly hydrolyzed to GDP once incorporated into microtubules. This allows microtubules to dynamically grow or shrink (dynamic instability)

Dynamic Instability

Refers to the characteristic behavior of microtubules in which their plus ends rapidly alternate between phases of growth (polymerization) and shrinkage (depolymerization) rather than existing in a stable, unchanging state.

This property arises from the balance between GTP-bound tubulin addition and GTP hydrolysis to GDP-tubulin, and it allows the cytoskeleton to quickly reorganize in response to cellular needs, such as during mitotic spindle formation or cell migration.

Microtubule Polymerization and Depolymerization

Growing microtubule is where tubulin dimer with bound GTP in the b tubulin is added onto the plus end of the microtubule or to the growing end of the microtubule. Addition of these dimers with GTP proceeds faster when the tubulin hetrodimers are high in concentration, than GTP hydrolysis by dimers.

Shrinking microtubule is where GTP hydrolysis is faster than the addition of now GTP tubulin dimers. Protofilaments (remember a microtubule is made of 13 of these long strands put next to one another to create a tunnel almost) containing GDP tubulin peel away from the microtubule wall and then the GDP tubulin is released to the cytosol.

Microtubule plus ends can be stabilized in cells by binding to certain proteins

Microtubules can technically depolymerize and polymerize from both the plus and minus ends when free (not bound) in a solution. However since microtubules in cells are generally stabilized at their base by y tubulin both growth and decay occurs only on the positive end at different rates.

What happens when microtubules are rendered stangant by drugs?

Microtubule dynamics are critical for cellular division and Taxol, a small molecule that binds to and stabilizes microtubules (preventing the dynamic instability crucial to functional microtubules) which ends up blocking cell division and is commonly used as a chemotherapy drug to treat breast cancer.

Squid Axoplasm Experiment

We know about microtubule function through an experiment that squeezed out axoplasm out of a giant squid axon which was then experimented on. From the experiment scientists derived that if ATP was removed, the organelles (cargo) did not move and if the ATP was replaced with non-hydrolysable ATP analog, cargo still did not move. These observations tell us that motor proteins use ATP hydrolysis to move cargo across microtubules.

Microtubule Motors (2 main types)

Microtubule motors position organelles in eukaryotic cells.

• Kinesin motors move ER tubules towards the edges of the cell, kinesin moves cargo toward the positive end of the microtubule

• Dynein motors move Golgi membranes towards the center of the cell, the Dynein moves cargo toward the negative end of the microtubule

Microtubule function in nerve cells

Nerve cells have long axons (up to 1 m (3 feet) in length in a human). Almost all biosynthetic processes for proteins, lipids, etc are made in the cell body, so these components need to be transported down to the end of the axon for growth and normal turnover – this is called outward (or anterograde) transport.

Material also has to be transported back from the end of the axon; this is called inward (or retrograde) transport. Microtubules extend along axons, and molecular motors perform these transport processes by carrying cargo along microtubules.

Motor Proteins and their Types (Microtubules)

Motor proteins move along microtubules using their globular heads.

Made of Globular heads attached to a tail and then that tail is attached to the motor protein cargo. The globular heads "walk" along the microtubule.

Two types of motor proteins are Kinesin and Dynein. Anterograde transport is mediated by a protein called kinesin, and retrograde transport is mediated by a motor called dynein.

Different motor proteins transport different types of cellular cargo (as Kinesins and Dyneins move in different directions and thus carry different products)

Kinesin and dynein both move possessively or step by step on the microtubule

Processive Motors

Describes an enzyme or motor protein's ability to carry out multiple rounds of catalysis or movement on a substrate without dissociating from it between steps.

They can take many successive steps along a microtubule or actin filament while remaining continuously bound, rather than releasing and rebinding after each step. This continuous engagement allows for efficient, sustained movement or activity over long distances, as opposed to non-processive enzymes that dissociate and must repeatedly find and rebind their substrate.

Coordination between heads allows processive movement

Examples of processive motors: Myosin V, Kinesin, Dynein

Structure of Kinesin and Dynein

Composed of two ATP binding motor domain "heads"

The coiled coil dimerization region is made of two alpha helixes wrapped around one another

The tail is what actually binds the cargo that the Kinesin/Dynein is transporting

How Kinesin and Dynein move along the microtubule

Kinesin moves "hand over hand" down a microtubule by coupling ATP hydrolysis to a conformational change

• The leading head (head more to the right of the microtubule) binds to ATP which causes a conformational change by propelling the trailing head by 16 nm or by 2 pairs of tubulin heterodimers

• The new leading head (used to be the trailing head) with bound ADP finds a new binding site on the microtubule

• The new leading head releases ADP and coordinately, the trailing head hydrolyzes ATP to ADP + Pi

• Kinesin hydrolyzes one ATP for each step along a microtubule

ATP vs ADP binding on Microtubule Motors

ATP vs ADP binding results in 2 key differences

• Affinity of head for Microtubule:

  • if ADP is bound, affinity is low

  • If ATP is bound, affinity is high (if no nucleotide, affinity is also high)

• Position of neck linker relative to motor head is different (conformational change)

Actin Filaments (aka Microfilaments)

Thin, flexible protein threads with a plus end and a negative end

Also called microfilaments, reversibly assembled from a polymer of actin, provide cells with their shape and contractile properties

They are made from the actin monomer which is bound to ATP rather than GTP like microtubule 

Actin Polymerization and Depolymerization

Actin can assemble into filaments with an end favored for assembly, the plus end, and one favored for disassembly, the minus end. However depending on concentration of actin monomers, they can both assemble and disassemble at the plus and minus end.

The ATP that is bound to actin is slowly hydrolyzed to ADP. But actin that is bound to ATP is added to the plus end and then that ATP is hydrolyzed and eventually actin monomers are usually lost at the minus end in a treadmill fashion at normal concentrations of actin monomers.

In cells, actin polymerization is controlled by actin nucleator proteins.

Actin Binding Proteins

Actin binding proteins mold actin filaments into functional structures (nucleating proteins, severing proteins, myosin is a big actin binding protein, cross linking protein, bundling proteins, etc. are all different examples of actin binding proteins)

These are proteins that modify and compliment actin

Myofibrils

A long, thread-like structure found inside a muscle fiber (muscle cell) that is responsible for muscle contraction. It is made up of repeating units called sarcomeres, which contain actin (thin) and myosin (thick) filaments.

Sarcomeres

Skeletal muscle contains muscle fibers in which myofibrils contain hundreds of repeating contractile units called ‘sarcomeres’. One sarcomere extends from one Z-disc to the next.

Sarcomeres are the contractile units of muscle.

• Each sarcomere is separated by z disks which connect them and they are made of 3 parts. One are the layered actin strands, then is the overlapping regions with both actin and myosin is present (where myosin strands connect to the actin), and then the middle region which is just composed to myosin strands.

• Sarcomeres are about 2.2 micrometers in length.

• Actin filaments extend in both directions from the Z-disc, with their plus ends at the Z-disc.

The movement of myosin heads on the actin filaments draws the Z-discs closer together, with the net result that the muscle contracts. Myosin and actin do not shrink during this process but the sarcomere does shrink.

Myosin Filaments (aka Thick Filaments)

Myosin molecules associate to form bipolar (like a double headed arrow) myosin filaments

Bundles of myosin II molecules assembled tail-to-tail, with their motor head domains projecting outward along the filament. Within the sarcomere, these heads bind actin (thin filaments) and use ATP hydrolysis to drive filament sliding, generating the force behind muscle contraction.

Myosin Families

All actin dependent motors belong to the myosin family and there are many different myosin genes (at least 14 different sub families of myosin genes have been identified)

Muscle myosin belongs to the myosin II subfamily.

Myosin V is not used in muscle tissue and functions similar to kinesins and dyneins but instead walk along actin filaments.

Myosin (II) Structure

Myosin II molecules are made of two heads connected to a coiled coil tail (two coiled alpha helix structures) and is about 150 nm in length

These myosin II molecules are attached to the bare region and this is what makes up the myosin II filament

How Myosin moves Actin

Each myosin head walks along the actin filaments due to a cycle of ATP binding and hydrolysis

• ATP binding causes the release of myosin from actin filament

• Then ATP hydrolysis causes conformational change, and the myosin head with the ADP + Pi tightly bound moves into a new position (cocked)

• The binding of the myosin head to a new site on the actin filament causes the release of the phosphate

• Phosphate release results in another conformational change, called the power stroke which generates a force and moves the actin filaments relative to the myosin filament

• ADP is released and myosin remains attached to the actin filament, ready for another cycle of movement

• We say this process is not processive because each motor head works independently

Thick filament

A thick filament is a protein structure in muscle cells is also known as a myosin filament and is made primarily of myosin. Myosin heads bind to and pull on actin during muscle contraction.

Thin filament

A thin filament is a protein structure made primarily of actin, along with regulatory proteins like troponin and tropomyosin. It interacts with myosin during muscle contraction, allowing the sarcomere to shorten.

Mutations and General Categories of Mutations

Permanent changes in the nucleotide sequence of DNA

There are various general categories of mutations

• Engineered (targeted) mutations: a permanent change in the nucleotide sequence of DNA intentionally introduced by scientists via molecular biology

• Natural (random) mutations: a randomly produced permanent change in the nucleotide sequence of DNA

  • Causes for random mutations: DNA polymerase replication errors, radiation, asbestos/chemicals, pollutants, environmental factors

Types of DNA Mutations

Single nucleotide (point mutations)

• Silent mutations: point mutations that do not alter the amino acid sequence

• Missense mutations: point mutations that lead to the incorporation of a different amino acid

• Nonsense mutations: point mutations creating a premature stop codon

Deletions: loss of a DNA sequence (1 deoxynucleotide or more)

Insertions: incorporation of an additional DNA sequence (1 deoxynucleotide or more)

• Frameshift mutations: a genetic change caused by the insertion or deletion of nucleotide bases in numbers that are not a multiple of three. Alters the order or set of 3 by which the mRNA is translated.

Chromosomal mutations: changes to the overall structure of a chromosome

Some points to remember about mutations

If a point mutation occurs in the original start codon in the DNA, the ribosome will just start translation at the next AUG it finds resulting in shorter protein sequences than intended. This can completely change the reading frame of the mRNA.

The amino acids that only have one codon cannot cause a silent mutation as there is no other redundant codon that can code for that same amino acid

Mutations in the primary sequence can interfere with protein folding

Mutations regarding K-RAS Small GTP Binding Proteins

K-RAS small GTP binding protein regulates cell growth, but the K-RAS small GTP binding protein's glycine at position 12 changes to valine which means that once the K-RAS small GTP binding protein cannot be turned off once activated. This leads to immense poliferation of cell growth signals which can contribute to cancer.

Around 30% of cancers have pepetually activated K-RAS small GTP binding proteins present.

We can add a competitive or noncompetitive inhibitor that either binds at the substrate binding site or at an allosteric site on the K-RAS GTP binding protein in order to solve some of the effects of the mutated version of the protein.

How Cells Deal with Unfolded Proteins

Protein degradation is performed via a ubiquitin proteasome system (UPS)

Proteins are marked by ubiquitin for degradation and ubiquitin chains stack and attach on proteins that are unfolded, malfunction, or at the end of their lifespan

The proteasome forms a chamber for protein hydrolysis. It contains a polyubiquitin binding site, a regulatory particle that seals the barrel of the proteasome from the top and the bottom, a proteolytic chamber that hydrolyzes the protein.

Proteasome is shaped like a barrel with a top that can be closed where the protein that needs to be degraded enters, and a bottom that can also be closed or opened to release the broken up protein in the form of amino acids. 

The regulatory particle can use the energy of ATP hydrolysis to unfold the bound proteins and then to transfer the unfolded peptide chain into the proteolytic chamber.

Proteasome Structure and Function

A large, barrel-shaped protein complex composed of a central 20S core particle, which houses proteolytic active sites, capped on one or both ends by a 19S regulatory particle that recognizes, unfolds, and translocates substrates into the core.

Its main function is to degrade proteins that have been tagged with ubiquitin chains, a process central to regulating protein levels, removing misfolded or damaged proteins, and controlling cell cycle progression.

This ubiquitin-proteasome pathway is a key counterpart to lysosomal degradation, targeting short-lived and regulatory proteins for controlled destruction.