BIOCHEM 2.8 - CYTOSKELETON, PUMPS, AND CHANNELS

• Study notes on Cytoskeleton, Pumps and Channels

Cytoskeleton Review

• A review of the cytoskeleton is provided with emphasis on structure and function
• The cytoskeleton is comprised of microtubules, actin filaments and intermediate filaments

Microtubules

• Forms polarized network for organelle and protein movement within the cell
• Hollow tubes, 25 nm with 15-nm lumen
• The protein subunit is tubulin, a dimer consisting of α-tubulin and ẞ-tubulin
• Microtubules maintain cell shape and motility
• Functions in chromosome movements in cell division and organelle movements
• Major component of cytoskeleton
• Provides cell shape and support
• Functions in SPINDLE formation during cellular division
• Tracks movement of material within the cell
• Provides for spatial organization of the cell and organelle movement
• Hollow rods are formed from α and β tubulin
• 25 nanometer fiber formed from 13 protofilaments
• Microtubules have orientation (+ and - ends)
• Bound GTP promotes growth; GTP hydrolysis frees dimers
• Found in Flagella and Cilia as a structural component

Actin Filaments

• Responsible for contractile and protrusive forces on cells
• Consists of two intertwined strands of actin
• Diameter of 7 nm
• The protein subunit is actin
• Maintains cell shape and changes in cell shape
• Responsible for muscle contraction, cytoplasmic streaming (plant cells), cell motility and cell division (animal cells)
• Two chains of actin (helix) create actin filaments
• 7 nm fiber with “pointed” and “barbed” ends
• Growth can occur at both ends but net growth tends toward "barbed" end
• Growth/assembly requires ATP; ATP hydrolysis frees monomers shortening the fiber
• Functions include cell shape, motility, cell division (cytokinesis), muscle contraction and cytoplasmic streaming
• Growth/assembly requires ATP; ATP hydrolysis frees monomers shortening the fiber
• Requires actin nucleators (Arp2/3 Complex)
• Frequently the nucleators must associate with nucleation promoting factors (NPFs) which enhances efficiency of polymerization
• Branching of actin filaments requires complexes such as Arp2/3

Intermediate Filaments

• Responsible for maintenance of cell shape
• Fibrous proteins coiled into cables
• Diameter of 8-12 nm
• Protein Subunits are one of several different proteins (such as keratins) such as vimentin, keratin and neurofilaments
• Maintain cell shape
• Responsible for anchorage of nucleus and certain other organelles and formation of nuclear lamina
• Functions include adding strength, bearing tesnsion, reinforcing cell shape and fixing positions of organelles and desmosomes
• Most diverse component of the cytoskeleton
• 8 to 12 nm thick INSOLUBLE fibers
• Seven amino acid pseudo-repeat sequence with specific localization of non-polar groups to form strip of hydrophobicity down axis of helix

Microtubule dynamics

• GTP binding triggers polymerization while GTP hydrolysis triggers depolymerization
• Microtubule associated proteins regulate growth, stability, and interaction with other cellular components
• Nocodazole interferes with polymerization while paclitaxel stabilizes microtubules

The Taxanes

• Family of compounds that stabilize microtubules by inhibiting depolymerization
• Each family member may impact microtubule associated proteins differently
• Paclitaxel reversibly binds the N-terminal 31 amino acids of the beta-tubulin subunit
• It shifts the equilibrium toward ASSEMBLY
• Microtubules inhibited with paclitaxel cannot depolymerize (lose catastrophe) and affects the number of tubulin heterodimers in microtubules

Pumps and Electro-chemical Gradients

• Electrochemical gradient = difference in electric potential over a distance across a membrane
• Membrane potential = resulting difference in voltage across a membrane
• Pumps are required for generation of electrochemical gradients that enable passive diffusion through an ion channel
• Polarized membranes can be summarized as a differential in charge where one side is positive and the opposite side is negative
• Depolarization shifts the internal negative charge such that the cell becomes less negative
• Cycles of polarization and depolarization = action potentials = transient and reversible

Sodium-Potassium Pumps

• Pumps and channels that regulate ion availability and localization are critical to function
• Wide range of activities are connected to sodium-potassium pumps including movement, entry of glucose into cells, and maintenance of low pH in the stomach
• Pumps are a combination of proteins (tissue specific FXYD proteins) combined with a catalytic α-subunit and obligatory β-subunit -multiple transmembrane segments are present

Proton Pumps

• Hydrogen ions can be released to corresponding environments via proton pumps
• Critical to lowering pH in those environments
• Example of Parietal cell
• Regulation of this process is crucial as chronic secretion of the strong acids by the pump causes gastric and duodenal ulcers
• Treatment: Omeprazole activated by the acidic conditions of the gastric lumen
• Omeprazole Modifies cysteine residues of proton pump thus inhibiting the pump
• Treatment of gastric and duodenal ulcers with a combination of antibiotics and omeprazole is the most effective due to role of H. Pylori in ulcer formation

Role of Calcium Channels in Movement

• Voltage-dependent calcium channels respond to membrane depolarization and thus generate cellular responses following changes in membrane polarity
• Increased cytoplasmic calcium from the sarcoplasmic reticulum in skeletal muscle leads to muscle contraction
• Calcium must be pumped out of the cytoplasm back to the sarcoplasmic reticulum for the muscle to relax
• The process in cardiac muscle is similar though those cells also utilize antiport systems for calcium movement linked to sodium-potassium pumps to regulate calcium concentrations and thus responses

Combined symport activity

• Glucose enters intestinal epithelial cells via a symporter called SGLT1 that is coupled to Na+
• Na+ enters cell bringing glucose along
• Sodium or potassium pumps create the gradient that lets sodium naturally diffuse back through the SGLT1 symporter
• Glucose is then transported back out of the epithelial cell via facilitated diffusion mediated by GLUT2

Glucose-Galactose Malabsorption (GGM)

• GGM is a very rare autosomal recessive disorder that can result in neonatal death if untreated and is also known as SGLT1 deficiency
• Pathogenic variation in the SCL5A1 gene that encodes SGLT1 includes missense mutations with variable frequency depending on the population
• Glucose and galactose are not properly absorbed and accumulate in the intestines
• Common symptoms include Neonatal onset of severe life-threatening watery diarrhea and dehydration, bloating, nausea, abdominal cramps, frequent urination, and rumbling sounds from extra gas in the intestines, and is clinically indistinguishable from disaccharidase deficiency
• Treatment includes avoiding foods with glucose or galactose and milk products, but they can consume fructose
• Normal growth and development can occur if caught and treated properly