4th week biol 4272 endocytosis for bb

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Endocytosis Lab protocol

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  • Endocytosis in Tetrahymena

    Week 4 BIOL 4272: Cell and Developmental

    Biology Lab Department of Biology and Biochemistry

    University of Houston

  • Hydrophilic head

    WATER

    Hydrophobic tail

    WATER

    phospholipid bilayer (with proteins too)

    The Plasma Membrane

    semi-permeable

  • The Fluidity of Membranes

    phospholipids in the plasma membrane can move within the bilayer

    most of the lipids, and some proteins, drift laterally

    rarely does a molecule flip-flop transversely across the membrane, spontaneously energetically unfavorable

    ATP-dependent flippases can move phospholipids from one layer to the other

  • (a) Movement of phospholipids

    Lateral movement (107 times per second)

    Flip-flop ( once per month)

    with flippases and ATP >> msecs

    The Fluidity of Membranes

  • Endocytosis

    In endocytosis, the cell takes in macromolecules by forming vesicles from the plasma membrane

    Endocytosis is a reversal of exocytosis, involving different proteins

    There are three types of endocytosis: Phagocytosis (cellular eating) Pinocytosis (cellular drinking) Receptor-mediated endocytosis

  • In phagocytosis a cell engulfs a particle in a vacuole

    The vacuole fuses with a lysosome to digest the particle

    Endocytosis

  • phagocytosis

    CYTOPLASM EXTRACELLULAR FLUID

    Pseudopodium

    Food or other particle

    Food vacuole Food vacuole

    Bacterium

    An amoeba engulfing a bacterium via phagocytosis (TEM)

    Pseudopodium of amoeba

    1 m

    Endocytosis

  • Tetrahymena after ingesting ink

  • In receptor-mediated endocytosis, binding of ligands to receptors triggers vesicle formation

    A ligand is any molecule that binds specifically to a receptor site of another molecule

    Endocytosis

  • RECEPTOR-MEDIATED ENDOCYTOSIS

    Receptor Coat protein

    Coated pit

    Ligand

    Coat protein

    Plasma membrane

    0.25 m

    Coated vesicle

    A coated pit and a coated vesicle formed during receptor- mediated endocytosis (TEMs)

  • In pinocytosis, molecules are taken up when extracellular fluid is gulped into tiny vesicles

    also called constitutive endocytosis

    Endocytosis

  • Constitutive Endocytosis

    can be imaged with fluorescent vital stains

    aminostyryl pyridinium molecules

    + >>

  • FM 1-43

    rapid resonance across ethylene bonds

    nonfluorescent in aqueous environment

    stabilized in nonpolar environments such as phospholipid bilayers

  • FM 1-43

    rapid resonance; weak fluorescence

    stable resonance; strong fluorescence

  • FM 1-43

    many studies subsequently use for a variety of dynamic membrane events like endocytosis in mammals and amphibians (must read review paper on Bb)

    initially used to study events at the neuromuscular junction synaptic vesicle recycling

    can be used in Tetrahymena

  • Overall Protocol for using FM 1-43FX to study Endocytosis in Tetrahymena

    test the effect of energy inhibition on endocytosis perform experiment in the presence of NaF and NaN3 F- inhibits glycolysis; N3- inhibits mitochondrial respiration (NO ATP will be made inside the cell)

    add FM 1-43 to cells take time points at 2, 20, 40 and 60 min fix each time point with glutaraldehyde capture images with a fluorescence microscope

    Endocytosis in TetrahymenaSlide Number 2The Fluidity of MembranesSlide Number 4EndocytosisSlide Number 6Slide Number 7Tetrahymenaafter ingesting inkSlide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16

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