Cellular and Molecular Biology Topics
Cytoskeleton and ECM
Functions of the cytoeskeleton include cell shape, tissue stability, motor proceses, intracellular transport, and cell division. There are three major types of cytoskeletal filaments: F-actin, intermediate filaments and microtubules. Each type of filament is made of protein monomers that bind either ATP or GTP. The energy molecule is hydrolized after polymerization.
F-actin filaments are about 8 nm in diameter and are made of G-actin monomers. These filaments are multifunctional and can be organized into a variety of structures that have an important role in supporting and strengthening cell and tissue structure, cell shape changes and migration.
Microtubules (MT) are about 25 nm in diameter and are made of tubulin monomers. They play a major role in intracellular transport of organelles and vesicles.
F-actin and microtubules form by addition of monomers to either end of the filament, and their monomers are subjected to treadmilling. Treadmilling occurs when subunits add to the (+) end and dissociate from the (-) end at about the same rate. Therefore, there is no change in length. This is related to the kinetics of addition and dissociation of the GTP and GDP-bound tubulin in microtubules or ATP-bound G-actin in F-actin. When both ends of the filament or tubule are exposed, polymerization occurs until the concentration of free subunits reaches a value above the critical concentration for the (+) and below the critical concentration for the (-) end. At such a steady-state, assembly at the (+) end equals net disassembly at the (-) end, resulting in no net gain but a net flux of sububits through the polymer, i.e. treadmilling.
Intermediate filaments (IF) are about 10 nm in diameter and are made of a variety of protein monomers, like keratins and neurofilaments. IFs are more stable and durable than F-actin and microtubules because they form rope-like structures, unlike the "treadmilling " polymers formed by actin and tubulin. Intermediate filaments form an elaborate network in cells that help individual cells resist mechanical forces like pulling and stretching, as well as support and strengthen cell and tissue structure.
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F-Actin
Actin monomers (G-actin) are polarized molecules, with a positive (+) “barbed end” and a negative (-) “pointed" end. The pointed end of G-actin-ATP binds the barbed end of another. The two remain attached after ATP is hydrolyzed.
Polymerization of monomeric G-actin to filamentous F-actin occurs in two stages. First there is a slow nucleation of a few G-actin monomers. After a critical mass is reached, elongation continues as more subunits attach at a faster rate at both ends of the filament. When actin filaments are growing, addition of monomers is favored at the barbed end. Eventually, the filament reaches a steady state in which elongation and dissociation of monomers occur at the same rate.
F-actin may be organized into either bundles or networks, providing support and strength to microvilli and adhesion structures. F-actin and myosin form belts around the apical surface of epithelial cells, actin as a “tension cable” to control cell shape. This joins cells together to form an “impermeable” barrier via transmembrane proteins at adherens junctions.
Some actin filaments are dynamic and their assembly/disassembly can drive cellular shape changes. The forces for shape change are provided by actin polymerization and myosin moving actin filaments (actin/myosin contraction). Actin depolymerization and repolymerization drive lamellapodia extension. Focal adhesions, a type of cell juction, attaches and detaches the cell to the basal lamina as it moves along.
Actin-binding proteins help organize filaments into different configurations, as well as regulate its assembly and breakdown. G-actin sequestrin proteins like thymosin prevent the monomers from assembling into fibers. Other proteins like profillin promote the assembly of F-actin. Gelsolin can severe actin filaments and at the same time cap the (+) end, preventing further polymerization. X-linking proteins like a-actinin and fimbrin assemble F-actin into bundles, while filamin and spectrin assemble F-actin into networks.
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Intermediate Filaments
Intermediate filament (IF) monomers have an alpha-helical core with four non-helical spacers common to all IF monomers. N- and C-terminus domains differ between monomers, and they project from IFs like wiskers.
The monomers coil around each other to form a dimmer with the N- and C-terminus aligned in parallel. Some IFs are homodimers, other heterodimers.
The antiparallel aligment of dimers forms tetramers, which then aggregate end-to-end to form a protofilament. The N- and C-termini project from the protofilament.
Pairs of protofilaments associate laterally into protofibrils. Lateral association of 4 protofibrils form 10 nm thick intermediate filaments. This makes for a very strong rope-like structure.
IFs are also very dynamic and will disassemble when phosphorylated. There are five major families of IFs: types I through V.
Type I IFs are made of acidic keratins and occur in the epithelia. Type II also accur in the epithelia but are made of basic keratins. Keratins forms network extending from the nuclear ernvelope to he plasma membrane.
Type III include several proteins that occur in a variety of tissues: vismentin in mesenchyme, desmin in muscle, glial fibillary acidic protein in glial cells and astrocytes, and peripherin in neurons.
Type IV occur in neurons and are made of either neurofilament (NF)-L, NF-M or NF-H.
Type V occur in the nucleus of all cells and are made of lamin A, lamin B and lamin C. Since some IFs are cell-type specific they are used for identification of tumor origin. Lamins form a lattice structure surrounding the nucleus. Phosphorylation regulates assembly and disassembly of lamins during mitosis, i.e. disassemble when phosphorylated.
IF’s distribute tensile forces and provide resistance of mechanical forces. An example is the role of keratin filaments in the epidermis, which is made of mostly the keratin filaments in skin cell desmosomes. Epidermal cells differentiate, condense and die, thus the keratin filaments remain intact providing a dead keratinized layer.
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Microtubules
Microtubules are cytoskeletal filaments composed of stable dimmers of alpha and beta tubulin monomers. The alpha subunit is irreversibly bound to GTP while the beta subunit binds and hydrolyze GTP. Hydrolysis occurs after incorporation of the monomer into a microtubule. Protofilaments form from end-to-end association of consecutive alpha and beta subunits. The protofilaments associate laterally into curved sheets until 13 protofibrils form a tubule.
Microtubules are polar filaments with one end of the filament having the beta subunit, the other the alpha subunit. The end with the beta subunit is the (-) end or slowest growing end. The end with the alpha subunit is the (+) or fastest growing end. If the rate of polymerization is faster than GTP hydrolysis, like when there is a large pool of GTP-tubulin, a “cap” of GTP-bound subunits forms. Microtubule assembly and disassembly occur preferentially (but not exclusively) at the (+) end. The ends of growing and shortening MTs look different: frayed if shortening, or thin and incomplete if growing.
MTs have distinct organizations depending on their localization and function. The 13 protofilament “singlet” forms cytoplasmic MTs. “Doublets” formed by one 13 protofilament ring joined to another 10 protofilament ring forms cilia and flagella. “Triplets” formed by one 13 protofilament ring joined to 2 other 10 protofilament rings form centrioles.
Microtubules
assemble from organizing centers (MTOC). For example, the centromere organize
cytoplasmic microtubules, and the basal body organizes MTs of cilia and flagella.
The overall orientation of MTs emanating from a MTOC is constant: (-) at the
MTOC and (+) in the periphery. Thus cargo is moved along in a specific direction,
depending on the motor protein.
In vivo assembly of microtubules is complex, characterized by a state of “dynamic instability” with oscillations between growth and shrinkage periods. Properties of MTs that contribute to the dynamic instability include the GTP cap favoring polymerization, faster depolymerization (~100X) at the (-) end than growth at the (+) end, and a rate of disassembly much higher than the rate of assembly. Since the (-) end is anchored at the MTOC, polymerization is hindered, therefore the MT must grow at the (+) end.
The GTP cap model has been proposed to explain dynamic instability of MTs. According to this model, a high concentration of free GTP-tubulin leads to rapid addition of monomers at the (+) end and a stable GTP cap. On the other hand, low concentration of free GTP-tubulin leads to more hydrolysis than addition at the (+) end and no GTP cap, making the (+) end more susceptible to hydrolysis.
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Motor Proteins
Motor proteins couple energy of ATP hydrolysis to motion (force) to move along filaments. Myosin moves along F-actin. Kinesin and dynein move along microtubules.
The
myosin motor couples movement of F-actin to ATP hydrolysis during contractile
processes. The myosins are a large family of proteins with similar domain structures.
The head domain binds F-actin and “walks” along the filament towards the (+)
end, pushing the filament. The tail domain has different functions, depending
on the myosin family member. In myosin I, the tail domain binds a vesicle membrane
for transport. In myosin II, the tail is a coiled alpha-helical structure that
polymerizes with tails of other myosin II molecules to form filaments. Both
myosin I and II have light chains near the head with regulatory function. Examples
of actin/myosin-driven shape changes include muscle contractions, cytokinesis
and migration.
Myosin must be “activated” in order to produce force. For example, Ca2+ activates it for muscle contraction. When skeletal muscle is stimulated, intracellular Ca2+ concentration increases and binds troponin, which in turn moves on the actin filament and exposes the myosin binding site. Myosin binds and releases the actin filament. Cycling of this movement causes actin filaments to slide pass myosin, shortening the saracomere. Each time that actin binds to actin requires one ATP to be hydrolyzed to ADP. The ADP is released when one stroke of the myosin head is completed.
While Ca2+ binds troponin in skeletal muscle, it binds a regulatory light chain kinase (MLCK) in smooth muscle. MLCK in turn phosphorylates myosin’s regulatory light chain (RLC), which the activates myosin. Another contractile preeocess, cytokinesis, is the contraction of a ring of actin/myosin resulting in formation of two daughter cells.
Another example of actin/myosin contraction is cytokinesis, when the contraction of a ring of actin/myosin results in the formation of two daughter cells.
Motor proteins move vesicles and organelles along microtubules that extend from the MTOC near the Golgi, with (+) ends pointing to the cell periphery. Kinesin dependent anterograde transport moves mitochondria, lysosomes and vesicles to the cell periphery. Dynein retrograde transport moves vesicles from the ER to the Golgi.
Motor proteins also move chromosomes along MTs during mitosis: kinesin pushes polar MTs and dynein pulls on astral MTs to help separate the chromosomes.
Both kinesin and dynein use the energy of ATP hydrolysis fror movement. The head domain binds MTs and hydrolyzes ATP while the tail domain binds cargo. There are two general types of dyenins: cytosolic and axonemal. Cytosolic dyenins are bound to cargo by a complex of MT binding proteins.
MTs
are important in cilia and flagella movements. Flagella are longer than cilia
and move in a sinusoidal wave. Cilia are shorter, have whip-like movements and
move synchronized over a whole area. Thye are both highly organized membrane-bound
bundles of MTs with very stable structure (in contrast with cytoplasmic MTs
which are very dynamic).
The bundle of MTs and associated proteins that form the core of cilia and flagella is known as an axonema.Both cilia and flagella have similar axoneme structure: 9 doublets arranged in an outer circle and two singlets in the center hub.
Specialized proteins in axonemes include ciliary dynein and nexin. Ciliary dynein is a motor protein attached to each of the 9 doublets, that moves adjacent MT doublets. Nexin is an X-linking protein between doublets. Cilia and flagella are anchored into a basal body, a MTOC that anchors the 9 sets of doublets and the 2 singlet MTs from the axoneme.
The dynein heads attached to the 13 filament tubule of one doublet walk along the 10 filament tubule of the adjacent doublet towards the (-) end. This forms a region of sliding next to the nexin-boun region that resist sliding, causing the tubule to bend. Regulating the timing and location of where the dynein motor is active, the bend propagates down the axoneme from the base (near the basal body) to the tip.
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Back to Basics: Physiology
Extracellular Matrix
The extracellular matrix (ECM) and basal lamina play a role in cell adhesion, force resistance, support, tissue structure, and serve as a track for cell migration and as a sink for signaling factors. The basal lamina is a flexible specialized ECM that surrounds and supports tissues and separate them form connective tissue. Cells secrete the components of the extracellular matrix: collagen, multiadhesive proteins and proteoglycans. Collagen fibers provide strength and resistance.
Proteoglycans are large complexes of glycosaminoglycans that branch from a linear protein core. Hyaluronin is the core of an extreamely large, negatively charged proteoglycan called aggrecan. These very large, highly hydrated molecules provide a viscous hydrated gel that resists compression forces.
Multiadhesive proteins bind components of the ECM to cell membrane receptors. Lamins and fibronectin are multiadhesive proteins that form multivalent linkages with collagen, heparin sulfate proteoglycans and integrins in the cell membrane.
Overall, these interaction link the ECM outside the cell to the cytoskeleton inside the cell through the transmembrane protein integrin.
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Cell Junctions
IFs are components of some cell-to-cell and cell-to-extracellular matrix connections critical to hold cells together in tisues. These components are the result of collaborative interactions between IFs, linker proteins and transmembrane cell adhesion molecules at specialized structures called junctions. Junctions serve to attach cels to each other and to the extracellular matrix and allow chemical and physical communication between cells to integrate their function. Cell adhesion molecules (CAMs) are transmembrane proteins that bind to the extracellular matrix or to another transmembrane proteins in the next cell. Linker proteins bind to the cytoplasmic tail of CAMs. Cytoskeletal intermediate filaments (or actin filaments) bind to linker proteins. Intermediate filaments are involved in desmosomes and hemdesmosome junctions. Desmosomes provede cell-to-cell connections. The CAM proteins in desmosomes are desmoglein and desmocollin, members of the cadherens family of transmembrane proteins. The linker protein is plakoglobin, a catenin. Desmosomal junctions look like “spot welds”. Hemidesmosomes are cell-to-extracellular matrix junctions found mainly on the basal surface of epithelial cells. They serve to attach cells to the basal lamina. The CAM in this case is a specific integrin, alpha-6-beta-4. The integrins in hemidesmosomes link the ECM to IFs in the cell through a plaque structure containing plectin.
In addition to desmosomes and hemidesmosomes, there are ther cell junctions that do not use intermediate filaments: tight (occluding) junctions, anchoring junctions and adherens junctions. Tight junctions are made of the transmembrane protein occluding. The extracellular domain of accludin forms very tight links with occludins of adjacent cells. Tight junctions form cell barriers and are important in establishing cell polarity (apical vs. basal surfaces).
Adherens
are actin juctions, either cell-to-cell (adhesion belts) or cell-to-matrix (focal
adhesions), similar to the desmosomes and hemidesmosomes fromed by IFs, respectively.
Adherens belts are formed when the transmembrane proteins caherens binds to
caherens on adjacent cells. A catenin complex serves to link cadherins and actin
filaments inside the cell.
Integrin is the transmembrane CAM in focal adhesions, while vinculin and talin link integrin to the intracellular actin filaments. Integrins are dimers of alpha and beta subunits from a large family of proteins. Adherens belts and desmosomes link membranes of adjacent cells providing strength and rigidity to the entire tissue. Focal adhesions mediate transient cell-to-ECM interactions. Different combinations of alpha and beta subunits determine the type of ECM component they will bind to. Cycles of adhesion and de-adhesion involve assembly and disassembly of focal adhesion sites enabling cell translocation.
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Advance Topics: Molecular Biology Lab
Diseases
Desmosomal failure occurs in skin diseases like pemphigus foliaceus and pemphigus vulgaris. These are autoimmune diseases, where antibodies are developed against desmosomal proteins. In pemphigus foliaceus, antibodies attack desmoglein-1, while in pemphigus vulgaris they attack desmoglein-3. This leads to breakdown of the desmosomal juction. Epidermolysis bullosa simplex (EBS) is a defect in keratin-14 that prevents intermediate filaments from forming, leading to epidermal blistering due to many dead basal cells and cell death from mechanical trauma.
Inmotile cilia syndrome (AKA Kartogener’s syndrome or primary cell dyskinesia) presents a clinical triad of symptoms: respiratory, situs inversus and infertility. Chronic bronchitis and sinusitis arise from retention of mucus due to nonfunctional cilia. Situs inversus occurs when certain organs (heart, liver) are rotated to their mirror image position. Infertility occurs because sperm flagella and fallopian tube cilia are defective. The defect is the loss of either one or both dynein arms in the axoneme.
Aquired ciliary defects are due to infections, environmental irritants or other pol;lutants. Injury leads to microtubule disorganization, changes in ciliary membrane integrity and remodeling of cellular structures in the airway.
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Advance Topics: Molecular Biology Lab
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