The cytoplasm of all eukaryotic cells is crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. This network, called the cytoskeleton, is a dynamic system, constantly assembling and disassembling. The cytoskeleton is made up of 3 major elements; microfilaments (“actin”) filaments, intermediate filaments, and microtubules.

Actin filaments (Microfilaments): 

Structure:

Actin filaments are the smallest component of the cytoskeleton ranging in size of about 7 nm in diameter. They are made up of actin and have a plus/barbed fast growing end, and a minus/pointed slow growing end. The addition of actin monomers occurs at the + end and is an ATP dependent process. Polymerization into filamentous f-action occurs when the concentration of globular g-actin is above a critical concentration.

The actin gene originated in the common ancestor of all life on Earth, as evidenced by the fact that bacteria, archaea, and eukaryotes all have actin molecules related structurally and functionally to each other. All eukaryotes have one or more genes for actin, and sequence comparisons have established that they are one of the most conserved gene families, varying by only a few amino acids between algae, amoeba, fungi, and animals. This conservation is attributed to constraints imposed by the interactions of actin with itself to polymerize, with motors and with a large number of regulatory proteins.  Humans have three genes for α-actin (muscles), one gene for β-actin (nonmuscle cells), and two genes for γ-actin (one in some smooth muscles and one in nonmuscle cells). Plants have 10 or more actin genes; some are specialized for reproductive tissues and others for vegetative tissues.(Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016). 

Actin is one of the most abundant proteins on Earth and the most abundant protein in many cells, from amoebas to human, often accounting for 10% or more of total protein. Its abundance is topped only by tubulin in brain and keratins in skin. Actin molecules in cells turn over very slowly, on the order of weeks in muscle cells. Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Actin is described as having four subdomains. The polypeptide winds from the amino terminus in subdomain 1 to subdomains 2, 3, and 4 and back to subdomain 1 at the carboxyl terminus. ATP binds in a deep cleft, interacting more strongly with subdomains 3 and 4, but also with residues in subdomains 1 and 2. Several proteins bind in a prominent groove between subdomains 1 and 3—and, hence, some call it the “target-binding groove” Actin monomers bind ATP or adenosine diphosphate (ADP) tightly, provided that either Ca2+ or Mg2+ is present in the buffer. One of these divalent cations associates with the β- and γ-phosphates of ATP, stabilizing its interaction with the protein (Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Actin-myosin interactions play crucial roles in the generation of cellular force and movement. The molecular mechanism involves structural transitions at the interface between actin and myosin’s catalytic domain, and within myosin’s light chain domain, which contains binding sites for essential (ELC) and regulatory light chains (RLC). (Thomas, ” Actin-Myosin Interaction: Structure, Function and Drug Discovery” Int. J. Mol. Sci. 2018, 19(9))

Functions/mechanisms of action:

Actin filaments are responsible for cellular movements such as contraction, crawling, “pinching” during division, and formation of cellular extensions. Essentially all cell motion is tied to the movement of actin filaments, microtubules, or both.  At the leading edge of a crawling cell, actin filaments rapidly polymerize and their extension forces the edge of the cell forward. Overall forward movement of the cell is acheived through the actin of the protein myosin, which is best known for its role in muscle contraction. Myosin motors along the actin filaments contract, pulling the contents of the cell toward the newly extended front edge. Cells crawl when these steps occur continucrously, with a leading edge extending and stabilizing, and then motors contracting to pull the remaining cell contents along. Receptors on the cell surface can detect molecules otuside the cell and stimulate extension in specific directions, allowing cells to move toward particular targets. 

Actin associated proteins perform many functions in arranging and stabilizing microfilaments including crosslinking (by filamen), bundling (the crosslinking of actin filaments into a parallel array as by ? actininand fimbrin), capping, severing and movement of structures along the fiber.

Contraction depends on the ATP driven sliding of highly organized arrays of actin thin filaments against arrays of myosin thick filaments. 

The zonula adherens/belt desmosome is an achorage junction that has a beltlike distribution and is associated with actin filaments.

Actin monomers polymerize spontaneously under physiological salt conditions with either or both monovalent and divalent cations in the buffer. Cations bind specific sites that promote interactions between subunits in the filament.  Pollard, “Actin and Actin-binding Proteins” Cold Spring Harbor Perspect Biol, 8/8, 2016).

Inhibitors: 

The drug, phalloidin, from the fungus Amanita phaloides (death cap), prevents the depolymerization of actin filaments. cytochalessins bind to the fast growing end preventing further addition of G-actin

Intermediate Filaments: 

Intermediate filamentsare intermediate in size compared to microfilaments and microtubules ranging with a diameter of about 10 nm. There are different types of intermediate filaments depending on the tissue where they are found.

The macula adherens/spot desmosome is an achorage junction which has a spot like distribution and is associated with intermediate filaments.

cytokeratins: are found in epithelia cells.

–Keratin is a filament protein which belongs to the intermediate filament protein family. Keratin fliaments are a major component of the cellular cytoskeleton. 

Keratin proteins are the predominant subtype of IFs in all epithelia and, given a total of 54 conserved functional genes and prtoeins, represents almost three-quarters of the entire IF superfamily in mammals. Kertin includes two types of IF sequences or families: type I, which number 28, and type II, which number 26. 

Keratins are a group of insoluble and filament-forming proteins. They mainly exist in certain epithelial cells of vertebrates. Keratinous materials are made up of cells filled with keratins, while they are the toughest biological materials such as the human hair, wool and horns of mammals and feathers, claws, and beaks of birds and reptiles which usually used for protection, defense, hunting and as armor. Keratins generally exhibit a sophisticated hierarchical structure ranging from nanoscale to centimeter-scale: polypeptide chain structures, intermediated filaments/matrix structures, and lamellar structures. (Fan, “Structure of Keratin” Methods Mel. Biology, 2021). 

vementin in connective tissue.

desmin in  

neurofilaments in axons and dendrites of nerves

nucleur lamins in nuclear lamina

Microtubules: 

Microtubules are formed from protein subunits of tubulin. The tubulin subunit is itself a heterodimer formed from two closely related proteins called alpha-tubulin (exposed at the + end) and beta-tubulin (exposed at the -end) tightly bound by noncovalent bonds. A microtubule is a stiff, hollow cylindrical structure built from 13 parallel protofilaments (long linear strings of subunits joined end to end that associate with one another laterally) They are larger than either microfilaments and intermediate filaments with a diameter of 25 nm. Addition of tubulin is a GTP dependent process unlike with the ATP dependent process of actin. 

Microtubules have one end attached to a single microtubule organizing center (MTOC) called a centrosome which is made up of 2 centrioles near the nucleus. 

Centrioles: are barrel shaped organelles found in the cells of animals and most protists. They occur in pairs, usually located at right angles to each other near the nuclear membranes. The region surrounding the pair is referred to a centrosome. The motor proteins kinesin and dynein move on microtubules and are involved with organelle transport. kinesins typically move from the cell body or centrosome toward the periphery of the cell towards the plus end of the tubule (anterograde) whereas dyneins typically move from the periphery toward the centrosome or minus end of the tubule (retrograde).

All eukaryotic cells must move materials from one place to another in the cytoplasm. One way they do this is using the channels of the ER, but material can also be moved using vesicles loaded with cargo that can move along the cytoskelton. For example, in a nerve cell with an axion that may extend far form the cell body, vesicles can be moved along tracks of microtubules from the cell body to the end of the axon. For components are reqired to do this: (1) a vesicle or organelle that is to be transported, (2) a motor protein that provides the eernergy driven motion, (3) a connector molecule that connects the vesicle to the motor moleule and microtubules on which the viscle will ride. The direction a vescile is moved depends on the type of motor protein involved and the fact that microtubles are organized with their plus ends toward the periphery of the cell. In one case, a protein called kinectin binds vesciles to the motor protein kinesin. Kinsin uses ATP to power its movement toward the cell periphery, dragging the vescile with it.Another set of vesicle proteins, called the dynactin complex, binds veiscles to the motor protein dyein which directs movement in the opposite direction toward the minus end inward toward the cell’s center. Dynein is also involved in the movement of eukaryotic flagella. 

Microtubules and the protein dynein also make up the motility structures, cilia and flagella. flagella are found on sperm and enable these cells to move. Cilia tend to be shorter than flagella. Both flagella and cilia have a distinctive 9 (doublet) + 2 arrangement of microtubules. Microtubules are also necessary in mitosis. 

A number of chemical agents also inhibits microtubule dynamics such as colchicine which binds to tubulin dimers preventing their assembly into microtubules.