Lecture 18: Intermediate Filaments and Microtubules 2024 PDF

In this lecture, we will investigate Intermediate Filaments and Microtubules The cytoskeleton is a network of three different filamentous proteins This image shows a cultured epithelial cell stained with a blue dye that labels all proteins – note that the cytoplasmic filaments represe...

In this lecture, we will investigate Intermediate Filaments and Microtubules The cytoskeleton is a network of three different filamentous proteins This image shows a cultured epithelial cell stained with a blue dye that labels all proteins – note that the cytoplasmic filaments represent a large proportion of the protein mass of this cell Proteins of the cytoskeleton function to provide different cell  types with their specific shapes Cytoskeletal filaments function to organize the cytoplasm Cytoskeletal networks can change their shape to produce large scale movements of cells and tissues The cytoskeletal network is composed of three networks Actin filaments, microtubules, and intermediate filaments occupy distinct, but overlapping, distributions in most cells The three filament types have different structures and functions (we’ll discuss in depth as we go) and different abilities to withstand forces All three cytoskeletal filament networks are biological polymers of their distinct monomeric subunits Filament polymerization (assembly) and depolymerization (disassembly) is regulated by the cell Biological polymers are different from chemical polymers Subunits associate reversibly by non-covalent interactions  This allows cells to polymerize and depolymerize cytoskeletal filaments to configure them to perform specific functions Intermediate filaments are the strongest filaments and are used by the cell to help withstand mechanical stress that is applied to the cell. Proteins such as plectin link intermediate filaments to other structures in the cell - forming a scaffold. Intermediate filaments come in a variety of types. There are intermediate filaments specific to the cytoplasm, and intermediate filaments (the lamins) that are nuclear. Each serve to provide structure and maintain mechanical integrity in these respective cellular compartments. All intermediate filaments have the same basic structure and assembly properties The IF monomer consists of an α-helical rod domain with globular N- and C-termini Unlike actin and tubulin, IFs do not bind to nucleotides All of the IFs share this organization but differ mostly in their globular domains Pairs of IF monomers dimerize  and dimers associate into tetramers with an anti-parallel orientation Note that each tetramer has two N- termini and two C-termini at each end: the structure of IFs, therefore, have no inherent polarity (unlike actin and microtubules) – if you flip it around 180 degrees, you get the same structure. Tetramers then associate laterally and twist together to form the final rope-like filament Filaments then elongate by addition of tetramers to either end (This is unlike actin and microtubules that have a polarized structure and a preferred end for polymerization) The central rod – coiled coil domain of IFs is conserved, but the flanking N- and C-terminal regions vary. These regions extend outwards from the filament and can mediate IF crosslinking (above structure). Structure below shows a single filament with terminal regions of each protein extending outwards. They type of IF expressed in a cancer cell can be used as a factor to identify the original tissue the cancer cells derived from, informing whether the cancer is metastatic or not. Keratins are intermediate filaments expressed in epithelial cells They form a mechanically durable network within the cytoplasm (A) epithelial cells stained for keratin (blue) and cell membranes (red) Note how bundles of keratins are indirectly connected to those in neighboring cells The sites of cell-cell interaction  at which keratins are attached to the membrane are called desmosomes This keratin/desmosome linkage functions to establish a continuous mechanical link from cell to cell throughout the entire sheet of epithelial cells Contributes to the mechanical durability of the entire tissue! Keratins also provide epithelial tissues with protection against lateral shear forces. If epithelial cells are plated on a flexible rubber sheet and this sheet is stretched, the cells are stretched but are protected from damage by the mechanical properties of their keratin networks If the same experiment is conducted with cells in which keratin has been targeted for knock out, the cells can no longer resist the stretching forces and they rupture in response Similar observations are made in animals that carry a mutation in keratin (A) shows a cross-section through the skin of a normal mouse (B) shows cross-section through the skin of a mouse mutant for keratin In the mutant, mechanical forces induce a splitting of the epithelial cell layers leading to  the formation of skin blisters (clear empty pocked between the cells) (C) Shows a schematic of how skin damage occurs in these mutants There are a number of skin blistering diseases in humans that can be traced to defective keratin mutations or to defects in the proteins that form desmosomes Nuclear lamins are IFs present in the nucleus of all eukaryotic cells Nuclear lamins form a tough network of filaments within the nucleus just underneath the nuclear membrane (B) shows a TEM image of the inner surface of the nuclear membrane; the nuclear lamins are clearly visible as a lattice- like 2-dimensional meshwork Lamins protect the nucleus from  mechanical forces Lamins also provide an attachment site for chromosomes during interphase and likely contribute to gene expression through mechanisms we do not yet understand Nuclear lamins are depolymerized during mitosis – this enables access of chromosomes to the mitotic spidle and proper chromosome segregation. The depolymerization is regulated by kinases. After mitosis, the nuclear lamins are dephosphorylated by phosphatases, which enables the filaments to reform. Evidence that nuclear lamins play an important role in gene regulation has come from studying a disease called progeria The image on the left is of young girl suffering from progeria Progeria afflicts people with symptoms of premature aging: it causes hair loss, wrinkled skin, atherosclerosis, blindness, kidney failure cardiovascular disease  People with this disease rarely survive into their teenage years Some forms of progeria are caused by mutation of a single amino acid residue in one of our lamin genes People with this mutation exhibit altered patterns of gene expression that could cause these symptoms How much of what we consider to be the ”normal” consequences of aging is due to changes in gene expression over our lifetime? Could insight gained from studying progeria patients tell us more about normal aging? Nuclei of progeria patients exhibit abnormal shapes that is consistent with a role for lamins in maintaining nuclear shape Although their dynamics are very different from actin and microtubules, IFs can be induced to assemble and disassemble The best-understood mechanism for IF dynamics comes from study of nuclear lamins When cells enter mitosis, nuclear lamins are disassembled to allow nuclear envelope breakdown so mitotic chromosomes can be  segregated on the mitotic spindle As cells enter mitosis, nuclear lamins are extensively phosphorylated causing them to dissociate into monomers At the conclusion of mitosis, lamins are dephosphorylated to allow the nucleus to reform in each daughter cell Cytoplasmic IFs are regulated in a similar manner The second cytoskeletal system we’ll discuss is the microtubule cytoskeleton Microtubules form a network of cytoplasmic filaments during interphase in most cell types Microtubule organization is altered throughout the cell cycle and they are the primary structural component of the mitotic spindle during cell division  Microtubules are also used by some cells to build motile cilia and flagella Microtubules are hollow cylinders composed of α- and β-tubulin The basic subunit (monomer) of microtubules are heterodimers of αβ tubulin – once synthesized there two proteins co-assemble into heterodimers and never dissociate When microtubules polymerize, tubulin heterodimers assemble in a head-to-tail manner into protofilaments  13 protofilaments associate to form the microtubule wall that is 25 nm in diameter Microtubules, therefore, have an inherent polarity with one end terminating in a β-tubulin (called the plus end) and one end terminating with an α-tubulin (termed the minus end) Microtubules are hollow cylinders composed of α- and β-tubulin The basic subunit (monomer) of microtubules are heterodimers of αβ tubulin – once synthesized there two proteins co-assemble into heterodimers and never dissociate When microtubules polymerize, tubulin heterodimers assemble in a head-to-tail manner into protofilaments  13 protofilaments associate to form the microtubule wall that’s 25 nm in diameter Microtubules, therefore, have an inherent polarity with one end terminating in a β-tubulin (called the plus end) and one end terminating with an α-tubulin (termed the minus end) This video shows a fruit fly cell expressing GFP-tubulin Microtubules exhibit a unique behavior called dynamic instability At any given time, individual microtubules grow and shrink randomly where growth is due to polymerization and shrinkage is due to depolymerization Even though individual microtubules grow and shrink, the total mass of  polymerized tubulin remains constant Both polymerization and depolymerization occur by tubulin addition and loss from the plus end, respectively Catastrophe: switching from growth to shrinking Rescue: switching from shrinking to growth Microtubules have a “cap” of GTP at their plus ends when they grow Soluble tubulin heterodimers bind to GTP Once they assemble into a growing microtubule, their polymerization activates a slow GTP hydrolysis mechanism within the heterodimer Eventually they hydrolyze GTP to GDP As long as new GTP-tubulins are  adding to the growing plus end, there is a cap of GTP-tubulin ahead of the older GDP-tubulin in the microtubule polymer If the rate of new tubulin addition slows, however, the wave of GTP hydrolysis “catches up” and the tubulins at the plus end hydrolyze GTP to GDP When GDP tubulin is exposed at the plus end, the interactions between neighboring tubulins is weak and this triggers a microtubule do depolymerize Thus, a growing microtubule must have a “cap” of GTP tubulins at its plus end to keep growing other wise it will shrink Microtubules exhibit dynamic instability. Microtubules can polymerize (primarily at their plus end) – this is called polymerization. During polymerization, GTP-bound tubulin is incorporated into the end of the lattice, forming a stable GTP cap structure. If the cap is lost – and/or the tubulin at the end of the lattice has undergone GTP -> GDP hydrolysis, the GDP-tubulin lattice is less stable, and prone to depolymerization. Thus, the microtubule can start to fall apart, or depolymerize as tubulin subunits dissociate and are lost from the lattice. The transition from polymerization to depolymerization is termed “catastrophe”. A depolymerizing microtubule can transition back to a polymerization phase through a transition termed “rescue”. Many microtubule associated proteins play roles in regulating dynamic instability. In this experiment, a red microtubule seed is used to nucleate microtuble growth form its plus and minus end The seed is created using a non- hydrolysable GTP analog: GMPCPP Green tubulin + GTP is then added, which polymerizes off the seed. Can you identify the plus and minus end. Also added are different microtubule associated proteins: EB1 and XMAP215 Look at how the microtubule dynamics are altered. Some microtubule associated proteins can bind specific regions of a microtubule. Some factors bind along the length of a microtubule (recognizing the GDP- lattice), while others recognize the minus end, or the plus end. EB1 recognizes the GTP-bound lattice, and thus localizes preferentially to the growing microtubule plus end. In this experiment, a red microtubule seed is used to nucleate microtubule growth form its plus and minus end The seed is created using a non- hydrolyzable GTP analog: GMPCPP Red tubulin (rhodamine-labeled tubulin) + GTP is then added, which polymerizes off the seed. Can you identify the plus and minus ends? What distinguishes them based on polymerization? Also added is the microtubule associated protein EB1 with EB1 labeled in green via a GFP-fusion tag. In most cells, microtubules do not grow randomly; instead they grow from a nucleating organelle called a centrosome Centrosomes are composed of two parts: 1. A central pair of centrioles that are barrel-shaped organelles that are themselves made of bundled triplet microtubules 2. A surrounding matrix of proteins (visible as an electron  dense “cloud” surrounding the centrioles in the micrograph on the left) called the pericentriolar material Centrioles act as a scaffold for the pericentriolar material The pericentriolar material is made up of numerous proteins and is the site of microtubule nucleation This video shows an experiment in which a centrosome was purified from cells and attached to a microscope coverslip to be viewed by time-lapse fluorescence microscopy Fluorescent tubulin and GTP were then added and microtubules can be observed to grow out from the centrosome like a seed Once they start to grow, the  microtubules exhibit dynamic instability, growing and shrinking Not that new microtubules only grow from the centrosome – it is acting as a nucleation site in this purified system just as it would inside a cell Microtubule polymerization from centrosomes requires another type of tubulin: γ-tubulin γ-tubulin in the pericentriolar material acts as the seed for new microtubule nucleation This arrangement leads to a radial organization of the microtubule network with the fast growing plus end oriented out into the cytoplasm The minus ends – are capped by their interaction with γ-tubulin and anchored at the centrosome in most cells Microtubule polymerization from centrosomes requires another type of tubulin: γ-tubulin γ-tubulin in the pericentriolar material acts as the seed for new microtubule nucleation This arrangement leads to a radial organization of the microtubule network with the fast growing plus end oriented out into the cytoplasm The minus ends – are capped by their interaction with γ-tubulin and anchored at the centrosome in most cells