Protein Subunits in Cytoskeletal Filaments

PROTEIN SUBUNITS IN CYTOSKELETAL FILAMENTS

Microfilaments

Actin

-Fungi, plant, animal

-Structural support, motility

MreB

-Rod-shaped bacteria

-Width control

Microtubules

Tubulin (alpha and beta)

-Fungi, plant, animal

-Structural support, motility, cell polarity

FtsZ

-Bacteria

-Cell division

Intermediate Filaments

Lamins

-Plant, animal

-Support for nuclear membrane

Desmin, keratin, vimentin, others

-Animal

-Cell adhesion

Mitochondrial Electron Transport and Oxidative Phosphorylation

MITOCHONDRIAL ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION

The electron transport chain is a system of linked electron carriers.

Three Characteristics:

1. By transferring their electrons to other substances, the NADH and FADH2 are reoxidized to NAD+ and FAD o that they can participate in additional substrate oxication reactions

2. The transferred electrons participate in the sequential oxidation-reduction of multiple redox centers (groups that undergo oxidation-reduction reactions) in four enzyme complexes before r

educing O2 to H2O.

3. During electron transfer, protons are expelled from the mitochondrion, producing a proton gradient across the mitochondrial membrane. The free energy stored in this electrochemical gradient drives the synthesis of ATP from ADP and Pi through oxidative phosphorylation.

*  Dinitrophenol (DNP) uncouples mitochondrial electron transport from oxidative phosphorylation by dissipating the proton gradient.

>  uncoupling by DNP decreases ATP production

Cell Biology: Cell Surface and Communication Part II

Cell Adhesion

• Two forms of interactions allow cells to aggregate into distinct tissues and provide a means for transfer of information between the interior and exterior of cells
  
• Cell-Adhesion Molecules (CAMs) source
  
• Allow cells in tissues to directly adhere to one another (cells of the same type, homotypic adhesion, or cells of different types, heterotypic adhesion)
• A CAM can directly bind to the same kind of CAM on an adjacent cell (homophilic binding) or to a different class of CAM (heterophilic binding)
• Cell-cell adhesions can be long lasting (nerve cells) or very weak (immune-system cells that roll along vessels)
  
• Four major families
• Cadherins, Ig superfamily, Selectins, Integrins
  
• Adhesion Receptors
  
• Adhere cells to the ECM

Cell Junctions

• Serve several functions
• Junctions impart strength and rigidity to a tissue
• Transmit information between the extracellular and intracellular space
  
• Control the passage of ions and molecules across cell layers
  
• Serve as conduits for the movement of ions and molecules from the cytoplasm of one cell to that of its neighbor
• Three major classes
• Anchoring Junctions and Tight Junctions
  
• Hold cells together into tissues
• Organized into three parts
• Adhesive proteins (CAMs and adhesion receptors)
• Adapter proteins (connect CAMs or adhesion receptors to cytoskeletal filaments and signaling molecules)
• Cytoskeletal Filaments
• Tight Junctions control the flow of solutes between cells forming an epithelial sheet
  
• Gap Junctions
• Permit rapid diffusion of small, water-soluble molecules between the cytoplasm of adjacent cells
• Contains clusters of channels between two plasma membranes separated by a gap

Cell Biology: Cell Surface and Communication Part I

Extracellular Matrix (ECM)

• The extracellular matrix is a jelly of proteins and polysaccharides in which the single cells of the simplest multicellular animals are embedded
• Cells produce and secrete these proteins and polysaccharides, creating their own immediate environment
• Components
• Proteoglycans = a type of glycoprotein that cushions cells and bind a wide variety of extracellular molecules
  
• Collagens = proteins that often form fibers; provide mechanical strength and resilience
  
• Fibronectin = soluble multiadhesive matrix proteins that bind to and cross-link cell-surface adhesion receptors and other ECM components
  
• Functions
• In animals, the extracellular matrix cushions and lubricates cells
• Provides mechanical support to tissues
• Provides a lattice through which cells can move
• Serves as a reservoir for many extracellular signaling molecules that control cell growth and differentiation
  
• Using different combinations of ECM components, the ECM can:
• strengthen a tendon, tooth, or bone
• cushion cartilage
• provide adhesion in most tissues
• provide the cell with environmental cues to know where it is and what it should do
• The Basal Lamina source
• A specialized, tough, sheetlike meshwork of ECM components that form a supporting layer underlying sheetlike cell layers and helps prevent the cells from ripping apart
• Have roles in regeneration after tissue damage and in embryonic development
• Helps organize cells into tissues and guides migrating cells during tissue formation
• Four ubiquitous protein components found in basal laminae
• Type IV collage = trimeric molecules with rodlike and globular domains that form a 2D netowrk
• Laminins = multiadhesive proteins that form a fibrous 2D network with type IV collage and also bin to integrins
• Entactin = rodlike molecule that cross-links type IV collage and lmainin and helps incorporate other components into ECM
• Perlecan = a large multidomain proteoglycan that binds to and cross-links many ECM components and cell surface molecules

• Cell-Matrix Adhesion source
• Adhesion receptors bind to various components of the ECM t0 meditate cell-matrix adhesions
• Responsible for directly or indirectly linking the CAM to the cytoskeleton (actin or intermediate filaments) and to intracellular signaling pathways
• Allows information to be transferred by CAMs and macromolecules in the ECM to which they bind
• Integrins
• Function as adhesion receptors to mediate many cell-matrix interactions
• are heterodimeric integral membrane proteins
• Members of the integrin family play important roles in adhesion and signaling in both epithelia and nonepithelial tissues
• integrins in hemidesmosomes help adhere cells to basal lamina
• some integrins participate in heterophilic cell-cell interactions in some blood cells
• Although they have low affinities for the ligands, binding of hundreds or thousands of integrins firmly anchor cells to the ECM
• Weak interactions also important to facilitate cell migration

Cell Wall source

• Plant Cell Wall
• A laminate of cellulose fibrils in a matrix of glycoproteins that completely coats the outside of the plant cell's plasma membrane
• Serves some of the same functions as the animal cell's ECM (although are composed of entirely different macromolecules and have a different organization)
• Connects cells into tissues
• Signals a plant cell to grow and divide
• Controls the shape of plant organs
• Has roles in controlling the differentiation of plant cells during embryogenesis and growth
• Structure
• Arranged into layers of cellulose microfibrils - bundles of long, linear, extensively hydrogen-bonded polymers of glucose in beta glycosidic linkages
• Cellulose microfibrils are embedded in a matrix of pectin and hemicellulose
• Layers of microfibrils prevent cell wall from stretching laterally
• Permeability of the cell wall is controlled largely by pectins
• No CAM plant homolog
• Adhesive-type proteins in plants are the wall-associat kinases (WAKs) and WAK-like proteins
  

Cell Biology: Technique: FRAP

Fluorescence Recovery After Photobleaching (FRAP)

Using FRAP, one can measure the rate at which membrane lipid or protein molecules move - the diffusion coefficient, as well as the proportion of the molecules that are laterally mobile.

source

You take a cell and designate a specific area of the membrane that you are interested in. The entire membrane is tagged with GFP and is fluorescent. You then take a laser and photobleach the area of the membrane that you designated. At that point, t=0, the area has no fluorescence. You then measure the amount of time the fluorescence returns to that area. When fluroescence returns to the area, you know that the membrane proteins have moved laterally through the membrane and been replaced with non-photobleached membrane proteins.

Protocol

1. Cells are labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein
2. Laser light is focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area
3. In time, fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is proportional to the fraction of labeled molecules that are mobile in the membrane. From this information, you can calculate the diffusion coefficient of the protein or lipid.

Cell Biology: Cellular Compartments Part IV: IMAGES

Cell Membrane System in Photos



Transport Processes in a Eukaryotic Cell























Properties of the two types of transport: Passive and Active

Channel and Carrier Proteins
Channel proteins are only responsible for passive transport, while carrier proteins are responsible for both passive and active transport

Carrier proteins bind one or more solute molecules on one side of the membrane, undergo a conformation change, and then deposit the solute molecules on the other side of the membrane




The glucose transporter, GLUT1, found in erythrocytes (a blood cell), is shown in its two conformations moving a glucose from the blood into the cell (the glucose is quickly phosphorylated within the cell so it cannot leave the cell)







Channel proteins tend to be ion channels and include three types which have both open and closed conformations (these channels are referred to as gated channels)

The Na+-K+ ATPase:
Required to maintain K+ high inside the cell and Na+ high outside the cell which is important to maintain the electrochemical gradient

The Na/K pump is usually made up of two alpha and two beta subunits. The alpha subunits are transmembrane proteins that have ATP binding sites on the cytoplasmic side. The beta subunits are located on the outer side of the membrane and are glycosylated. This figure shows the protein in the E1 conformation, meaning it is open to the cytosol. When Na+ ions bind, the protein changes to the E2 conformation, which opens to the outside. As shown in the figure, the pump is inhibited by oubain.

Cell Biology: Cellular Compartments Part III

MITOCHONDRIA AND CHLOROPLASTS

• Overview of Mitochondria source
• Main site of ATP production during aerobic metabolism are very large in size
• Outer membrane is about half lipid and half protein, contains porins that render the membrane permeable to molecules having molecular weights as high as 10,000 (a similar membrane to that of gram-negative bacteria)

• The inner membrane is less permeable is 80%protein, 20% lipid and forms cristae, or infoldings, that protrude into the matrix
• Degrades glucose to CO2 and H2O to produce 28 molecules of ATP (2 more molecules of ATP are produced in the cytosol)
• Overview of Chloroplasts source
• 
• Carry out photosynthesis
  
• Are surrounded by a double membrane and contain a network of internal membrane-bounded sacs

• Biogenesis of Mitochondria and Chloroplasts
  
• Grow by the incorporation of cellular proteins and lipids, and new organelles form by division of preexisting organelles - both processes occurring continuously during the interphase period of the cell cycle
• Evolution source
  
• Hypothesis that mitochondria and chloroplasts arose by the incorporation of bacteria into ancestral eukaryotic cells, forming endosymbiotic organelles
• Endocytosis of a bacterium by an ancestral eukaryotic cell would generate an organelle with two membranes, the outer membrane derived from the eukaryotic plasma membranes and the inner one from the bacterial membrane
• The F1 subunit of ATP synthase, localized to the cytosolic face of the bacterial membrane, would then face the matrix of the evolving mitochondrion or chloroplast membrane, such as occurs during development of chloroplasts in contemporary plants, would generate the thylakoid vesicles with the F1 subunit remaining on the cytosolic face, facing the chlorplast stroma
  
• Share with bacteria many proteins of similar sequences, including some involved in membrane translocation
  

Cell Biology: Cellular Compartments Part II

NUCLEUS

• Structure
• Nuclear Envelope made up of two phospholipid bilayer membrane
• Inner nuclear membrane is around nucleus itself
• Outer nuclear membrane is continuous with the rough ER
• The two membranes fuse at nuclear pores
• nuclear pores = ringlike complexes composed of specific membrane proteins through which material moves between the nucleus and the cytosol
• Nuclear Matrix
• Made of fibrous proteins called lamins which form a TD network along the inner surface of the inner membrane, giving it shape and apparently binding DNA to it.
  
• Functions
• Replicates DNA
• Syntehsizes rRNA, tRNA, and mRNA

Cell Biology: Cellular Compartments Part I

Cellular Membrane Systems

• Structure
• Bacterial, fungal, plant cells have a cell wall that gives their membrane rigidity
  
• Cell membrane restricts flow of molecules into and out of the cell
  
• Eukaryotes: Membranes separate cell into compartments, organelles
• 2 Layers of phospholipid molecules (hydrophilic and hydrophobic end)
• Lipids (i.e. cholesterol) and proteins are inserted into phospholipids
• Molecules making up the membrane can float sideways, giving the membrane a fluid character allowing shape-change and movement (but attachment to proteins w/in cell can stop movement
• Lipid Composition of the Cell Membrane
  
• Phospholipids spontaneously form bilayers
• Hydrocarbon chains form the hydrophobic core
• Bilayer structure maintained by hydrophobic and van der Waals interactions btwn lipid chains
• Always a cytosolic face and an exoplasmic face
• Phosphatidylcholine, the most abundant phospholipid in the membrane
• Transport
• Membrane Transport Proteins
• Definition
  
• permit passage of nutrients into the cell and metabolic wastes out of it
• Keeps the cytosolic conc of K+ much higher than that of Na+ in the cell (i.e. K+ much higher in the cell, Na+ much higher in the blood)
  
• maintain the proper ionic composition and pH (~7.2) of the cytosol
• are transmembrane proteins containing multi membrane-spanning segments (i.e. alpha helices)
• 3 Types of Membrane Transport Proteins source
• ATP-powered pumps (a.k.a. pumps)
• ATPases that use the energy of ATP hydrolysis to move ions or small molecules across a mem against a chemical conc gradient, electric potential, or both
• Process is called active transport and an ex. of a coupled chemical rxn (i.e. transport of ions 'uphill' against an electrochemical gradient is coupled to the hydrolysis of ATP)
• Process is energetically favorable
• Four classes of ATP-powered transport proteins
• P-class pump: Plasma membrane of plants, fungi, bacteria (H+ pump)
• Possess two identical catalytic alpha subunits that contain an ATP-binding site and one of the alpha subunits is phosphorylated
  
• :Plasma membrane of higher eukaryotes (Na+/K+ pump which maintains the low cytosolic Na+ and high cytosolic K+)
• F-class and V-class ion pumps
• contain several different transmembrane and cytosolic subunits and only transport protons
• F-class power the synthesis of ATP from ADP and Pi by moving protons from the exoplasmic to the cytosolic face of the membrane down the proton electrochemical gradient and are very important in ATP synthesis
• ABC superfamily (ATP-binding cassette)
• specific for a single substrate which could be ions, sugars, a.a.s, phospholipids, peptides, polysaccharides, or proteins
  

• Channel Proteins
• transport water or certain ions and hydrophilic small molecules down their conc or electric potential gradients.
• Protein-assisted transport referred to as facilitated diffusion
• Form a hydrophilic passage way across the membrane through which ions, etc. move through, single file, at a rapid rate
• non-gated channels: open much of the time
• gated channels: what most channels are, open only in response to specific chemical or electric signals
• Transporters (a.k.a. carriers) source
• Maintain ionic gradients across cellular membranes
  
• Three types:
  
• Uniporter transport a single type of molecule down its con gradient (i.e. glucose and amion acids
  
• Antiporters and symporters (a.k.a. cotransporters) couple the movement of one type of ion or molecule against its conc gradient w/ the movement of one or more different ions down its conc gradient
  
• Passive (simple) diffusion
• Doesn't require energy b/c moves down conc gradient
  
• Gases (O2, CO2) & small uncharged polar molecules (urea, EtOH)
• To measure rate, add a small amt radioactive material to one compartment & meas its rate of appearance in other compartment
• Larger the partition coefficient, faster it will diffuse across a mem
  
• Osmosis = water moves semipermeable mem from low solute to high solute conc until both sides equal
• isotonic solution= solute conc of sol equal to the conc inside cell
  
• hypotonic solution = solute conc of solution is LESS than inside the cell
• hypertonic sol = solute conc of solution is MORE than inside the cell
• Features Distinguishing Uniport from Passive Diffusion
• Rate of facilitated iffusion by uniporters is higher than passive diffusion
• B/c the transported molecule never enters the hydrophobic core of the bilayer, the partition coefficient is irrelevant (doesn't have to be large to be fast)
• Transport occurs via a limited number of uniporter molecules rather than throughout the entire bilayer. As a result, there is a max transport rate Vmax that is reached when the conc gradient across the mem is very large and each uniporter is working at its max rate
• Transport is specific; each uniporter transports only a single species of molecule or a single group of closely related molecules
• The GLUT1 Uniporter source

• glucose transporter found in plasma membrane of erythrocytes (no nucleus or other internal organelles making it easy to purify transport proteins)
• GLUT1 expressed by most mammalian cells since most mammalian cells use blood glucose as the major source of cellular energy
• Alternates between two conformations as seen on figure
• Isomeric sugars D-mannose and D-galactose, which differ from D-glucose is the configuration at only one carbon atom, are transporter by GLUT1 @ measurable rates; however, the Km for glucose is much lower than for the other sugars, meaning GLUT1 is still fairly specific for glucose
  

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