Cytoskeleton: Cell Shape and Movement

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Lecture 14: Cytoskeleton, Part I

• Cytoskeleton functions include maintaining cell shape, cell adhesion, structural support, cell movement/division, and intracellular movement of proteins/organelles.
• Intermediate filaments provide mechanical strength to cells, enabling them to withstand stretching.
• Intermediate filaments form a network around the nucleus, extending to the plasma membrane.
• Intermediate filaments are rope-like fibers made of elongated proteins with lateral interactions between subunits.
• Intermediate filament subunits are elongated filaments (homodimers).
• Homodimers bind antiparallel to form a staggered tetramer (protofilament).
• Protofilaments connect with 7 other tetramers.
• Nuclear lamins are a type of intermediate filament subunit forming the nuclear lamina beneath the inner nuclear membrane.
• Nuclear lamins are phosphorylated by kinases during mitosis, causing nuclear membrane disassembly.
• Dephosphorylation of lamins by phosphatases at the end of mitosis allows them to reassemble into the nuclear lamina
• Microtubules provides tracks for transporting vesicles, organelles, and cell components
• Microtubules form the mitotic spindle for chromosome segregation during mitosis
• Microtubules facilitate cell movement via flagella and cilia.
• Microtubules consist of hollow, polarized cylinders of α- and β-tubulin
• Intermediate filaments are flexible, non-polar fibers providing structural support, but not for transport.
• Microtubules and actin filaments are dynamic due to polarity at their ends.
• Intermediate filaments are stable due to identical ends creating no polarity.
• Intermediate filaments are made of subunits having lateral interactions.
• Microtubules are composed of globular subunits with fewer lateral interactions.
• Tubulin is a heterodimer of alpha and beta subunits.
• Beta subunit can bind and hydrolyze GTP to GDP.
• Alpha subunit can bind GTP but cannot hydrolyze it.
• The subunit of microtubules is a heterodimer of alpha and beta tubulin.
• A protofilament consists of tubulin dimers (subunits) bound head-to-toe in a straight line, giving it polarity.
• The protofilament plus end contains the Beta subunit.
• The minus end has the alpha subunit.
• Microtubules are hollow structures composed of 13 parallel protofilaments.
• The plus end grows quicker, indicating a high rate of polymerization,
• The minus end indicates a slower rate.
• GTP hydrolysis in microtubules is crucial for dynamic instability.
• GTP hydrolysis results in rapid polymerization and depolymerization by changing the shape of subunits/protofilaments.
• A GTP cap is region at the growing end of a microtubule where tubulin dimers have GTP.
• The GTP promotes polymerization, but when hydrolyzed to GDP in older subunits, microtubules destabilize
• If tubulin-GTP is added before complete hydrolysis, the microtubule can rescue itself and continue growing.
• Dynamic instability is the rapid switching between microtubule growth and shrinkage.
• This is due to the addition and loss of tubulin dimers at the microtubule plus end.
• A GTP cap regulates this with stability when GTP is present, but rapid depolymerization occurs of GTP is lost.
• Microtubules elongate when tubulin dimers with GTP bind to the plus end.
• Over time, GTP gets hydrolyzed to GDP, weakening the structure.
• A high tubulin-GTP concentration in tests are required for rapid polymerization because the rate of addition is faster than the rate of GTP hydrolysis.
• When concentration decreases, the rate of addition decreases and the GTP cap turns into a GDP cap.
• Microtubules need a high concentration of subunits to grow in a test tube.
• Cells regulate microtubule growth even at low subunit concentrations given regulatory factors to aggregate rapidly.
• Gamma-tubulin ring complexes (γ-TuRCs) serve as nucleation sites for microtubule growth, located on the centrosome surface.
• Gamma-tubulin ring complexes mimic the (+) end and template the 13 protofilaments.
• Cells regulate the number of microtubules using nucleation sites.
• Tubulin-GTP concentration determines the rate of growth.
• Kinesin carries organelles and vesicles toward the plus end of microtubules.
• Dynein also carries organelles and vesicles, however it transports to the minus end of microtubules.
• In neurons, kinesin transports cargo from the cell body to the nerve terminal
• Dynein transports cargo from the nerve terminal back to the cell body.

Lecture 15: Cytoskeleton, Part II

• Functions of actin filaments include: microvilli increase intestinal cell surface area, changing cell shape, cell motility, and contractile ring formation during cell division (cytokinesis).
• Actin is the subunit of actin filaments that are asymmetrical monomers with a plus and minus end binding to ATP.
• Two protofilaments of actin monomers form a helix forming the actin filament.
• Two protofilaments form a helix.
• Actin filaments are helical polymers with a plus end (fast growing) and a minus end (slow growing)
• Actin monomers hydrolyze bound ATP to ADP following incorporation into the actin filament.
• Hydrolysis decreases the stability of the actin subunit.
• Polymerization depends on the amount of actin monomers available.
• The three steps of actin polymerization in a test tube are: nucleation (lag phase), elongation (growth phase), and steady state (equilibrium phase).
• Nucleation is when concentration of actin monomers is high and an initial aggregate of monomers (critical nucleus) will form
• Elongation is when initial aggregates start rapidly growing filaments by subunits attaching to the initial aggregate, decreasing subunit concentration.
• Steady State occurs when concentrations of subunits decline and filaments reach steady state called 'treadmilling' when the rate of addition equals the rate of dissociation.
• Adding preformed actin fragments to a test tube with actin monomers and ATP increases the rate of polymerization.
• There is NO LAG PHASE. This actin polimerization occurs in cells
• Treadmilling involves simultaneous gain of monomers at the plus end and the loss of monomers at minus end.
• Dynamic instability involves rapid switch from growth to shrinkage only at the plus end.
• Actin polymerization is regulated by proteins, such as Arp complex, profilin, and thymosin.
• Thymosin and profilin regulate actin subunits and some regulate actin filaments.
• The Arp complex nucleates actin filament growth by aggregating two actin monomers.
• The Arp complex binds to the minus end of 2 growing actin monomers.
• The aggregates form actin filaments and producing an interconnected network.
• Thymosin allows cells to have a high concentration of actin subunits by preventing assembly.
• Profilin binds to actin and promotes assembly, actively determining when/where polymerization occurs.
• The Arp complex makes branched actin networks driving membrane protrusions.
• Profilin enhances actin polymerization via ATP-actin monomer supply.
• Thymosin regulates monomer availability and coordinates rapid actin assembly for membrane extension.
• Myosin II interacts with actin filaments allowing important muscle contraction.
• Myosin II motor proteins have common features but have a variety in different cell types.
• Myosin II moves towards the plus end of actin filaments.
• The myosin head domains bind to ATP and actin, possessing ATPase activity.
• The neck domains act as lever arms for movement.
• The tail domains allow monomer subunits to dimerize giving aggregate with other dimers to form myosin filaments.
• Myosin II in unique because it is the only myosin that can form bipolar filaments.
• The tail domains of Myosin II bind each other making orientations, producing movement in opposite directions.
• Formin is an actin binding protein, nucleating unbranched actin filaments by binding to the plus end.
• Both Arp and formin promote nucleation.
• Formin allows progressive elongation at the barbed end.
• Arp complexes do nucleation binding to the minus end and attaching to existing filaments.
• Sarcomeres are the basic contractile unit of muscle fibers and contain repeating actin and myosin filaments.
• Actin filaments are anchored at the Z disc by their plus ends, while tropomodulin is at the minus end.
• Myosin II filaments lie in the middle, interacting with actin to shortening distance between Z discs contraction.
• Tropomyosin blocks myosin-binding sites on actin filaments, preventing contraction in a resting muscle.
• Troponin regulates this inhibition by binding to Ca2+ levels.
• Calcium binding causes conformational change causing tropomyosin to shift, exposing myosin-binding sites on actin and allowing muscle contraction.
• Myosin relies on atp to promote sarcomere contraction.
  • Myosin lacking ATP or ADP tightly binds as calcium comes back
  • ATP then binds to the myosin causing detachment from the subunit.
  • ATP hydrolyzes to change myosin position
  • Myosin then binds weakly at new filament - Release of ADP causes it to bind tightly again, and repeat.
• Sarcomeres shorten due to actin sliding past myosin during contraction, bringing Z discs closer.
• A sarcomere is a contractile unit with a myofibril, found in the skeletal muscle cell.
• Myofibrils comprise repeating sarcomeres lined end-to-end, with multiple myofibrils spanning muscles.

Lecture 16: Cell Adhesion

• Epithelial tissues (lining of intestinal cells, skin) are tightly bound by cell-cell junctions, have very little EC matrix, and are anchored to the basal lamina.
• The basal lamina is a specialized extracellular matrix providing binding sites for cell-matrix junctions.
• Connective tissues are secreted from sparsely distributed cells form ECM, and reside below the basal lamina and often contains blood vessels.
• Epithelial tissues have both cell-cell and cell-matrix junctions.
• Anchoring junctions use the cytoskeleton in order to connect adjacent cells or the matrix, increasing the mechanical strength of tissue.
• Tight junctions do not act as anchoring junctions.
• Adheren junctions use actin filaments; desmosomes use intermediate filaments and both are anchoring junctions.
• Gap junctions are involved in cell communications instead of anchoring junctions.
• The plasma membrane is spanned by Transmembrane adhesion proteins (TM).
• One end binds the cytoskeleton, the other binds to a structure outside.
• Adhesion can be homophillic or heterophillic.
• Adapter proteins link TM adhesion proteins to the cytoskeleton
• They bind to TM adhesion proteins, as well as actin or intermediate filaments.
• Cadherins mediate cell-cell junctions between proteins.
• Calcium gives shape to catherins.
• Catherins allow cells of the same tissue to adhere to each other preferentially through allowing homophilic binding.
• Catenin is an adapter protein, linking cadherin and actin filaments
• Membrane protrusions from actin polymerization (Arp complex) start the adheren junctions.
• After contact, cadherins and catenins cluster, forming adheren junctions.
• Desmosomes use different cadherin proteins that link to intermediate filaments of actin filaments.
• Adheren junctions are assembled and disassembled in response to cell signals.
• Desmosomes are more stable and are found in tissues that experience mechanical stress.
• Gap junctions allow channels to create between the cells using connexin as a TM protein.
• Glycosylated proteins and polysaccharides are found in the EC matrix and secreted by cells.
• Varying composition for hard or soft structures.
• GAGs are repeating disaccharides with spongy material for compression.
• GAGs when bounded to proteins creates proteoglycans.
• Collagen strengthens and organizes the matrix through aggregates for connective tissues.
• Fibronectin is a scaffolding protein with binding sites for adhesion and matrix proteins.
• ECM has both hard bone and soft cartilage by components.
• Collagen is for support found in ECMs.
• Integrins are TM adhesion proteins like catherins.
• They can attach to both filaments, but instead of catherin homophillic binding, they bind to fibronectin (heterophillic)
• Integrins in call matrix junction through collage bind to ECM fibronectins.
• Hemidesmosomes and actin-linked cell-matrix junctions connect epithelial intgrins to collagen and filaments.

Lecture 17: Cell Signaling, Part I

• Signal transduction converts one signal form to another.

• Ach signals elicits different response dependong binding to receptors of different cells

• The cell is transduced by how the signal is produced.

• Cell signaling happens in three phases:

  • Reception: signals either enter cells or bund receptors in membrane.
  • Transduction: signals are converted from the inside, and then amplified signal and recepetors - Response: external signals allow activiation of target proteins and can feedback genes as needed.

• Primary transduction converts TM receptor to intracellular signal by intracellular domain phosphorylation of RTK or GPCR acrivation,

• Essential regulation is achieved by a cell by the use of molecular "on" and "off" switches allowing biological and response with certain stimuli.

• Control systems and processes such as differentiation, growth, etc also help.

• GTPase and Protein Kinase help during the kinase cascade and cycle.

• Effector proteins excute cellular signaling event.

• Some single pass TM receptors are used as stimulus molecules with enzyme or associated in cells.

• Proteine kinase is important to recptor.

• Typically phosphoryalte one amino acids by cytoplasmic kinase.

• Kinases typically phosphorylate one amino acid causing self assembly after binding.

• Receptor tyrosine kinases (RTKs) binds to tyrosine amino acids with intracellular kinase.

• the binding cause kinase activation which generates docking sites amplifiying the extracellular signal.

• EGF receptors are only active as a monomer, where it bind and dimerize leading to down signaling.

• Grb2 is a scaffolding protein that binds phoshyrated tyrosine on activated RTK allowing GEF to activate Ras protein..

• Ras is the GTP binding protein which anchors to lipid bilayer transferring TM recepor to plasma.

• When signal molecule binds the recptor dimerize causing Grb2/Sos recruitment.

• Ras GEF then allows Ras to activates Raf. It then phosphorylates Erk which activates a transciption fator to gene expression.

• MAPs (Erk) then go on to active the effector protiend to respond and allows cell dividion.

• Mutations can lead to tumors by having EGFrs be active even without needed causing constant division.

• increasing EGFR numbers lead to excess dividing and growth factor.

• HER2 are human growth division factor.

• increased division can come from cell division with factor absent, or mutation.

• TkIs bind intracellular.

Lecture 18: Cell Signaling, Part II

• GPCRs are receptors activating the activation of membrane when linked to proteins or channels of plasms membrane through lipids, neurotransmitters etc.
• They have different units, but some role allow others to activate as the unit binds ot GTP.
• Alpha subunits get attached to gamma subunits allow transduction of the units to swap on TM receptors when activated from the alpha.
• From here, there are multiple different path ways of the proteins getting created.
• Second Messanger is made from being an actvated signal which in the cell and allow increases after binding.
• Cyclic amp (cAMP) is the 2nd messanger when ATP creates and binds to alpha GTP by adenylyl.
• cAMP triggers pKA activitity to allow activity and it allow for protein transfer.
• CREB then binds for alteraion of gene exprression.
• G proteins the activates the cyclades activate the beta and gamma to phosphplipaase activation.
• These messengers then activate protein for expression and increase.
• Signalling can be by neuronic, paracrine or endocrine.
• Endocrine relies on blood.
• Neuronal relies on impulses that stimulate through brain
• Paracrine relies on extracellular matric for a signal to shift for survival.

Paper 2: Ligand-Induced Redistribution of a Human KDEL Receptor from the Golgi Complex to the Endoplasmic Reticulum

• ERD2 is a retrograde protein main for golgi found with kDELS to allow the protein to enter the ER in inactive.
• lysososmes are found in secretions whether from breast or saliva. kDEL keeps proteins inside ER instead.