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Endocytosis in

Tetrahymena

Week 4 BIOL 4272: Cell and Developmental Biology Lab Department of Biology and Biochemistry University of Houston

The Plasma Membrane


semi-permeable

phospholipid bilayer (with proteins too)

Hydrophilic head Hydrophobic tail

WATER

WATER

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

The Fluidity of Membranes

Lateral movement (107 times per second) (a) Movement of phospholipids

Flip-flop ( once per month) with flippases and ATP >> msecs

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

Endocytosis
In phagocytosis a cell engulfs a particle in a vacuole The vacuole fuses with a lysosome to digest the particle

Endocytosis

EXTRACELLULAR FLUID

CYTOPLASM
Pseudopodium

1 m

Pseudopodium of amoeba

Food or other particle Food vacuole

Bacterium Food vacuole An amoeba engulfing a bacterium via phagocytosis (TEM)

phagocytosis

Tetrahymena
after ingesting ink

Endocytosis
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

RECEPTOR-MEDIATED ENDOCYTOSIS

Coat protein Receptor Coated vesicle

Coated pit Ligand

Coat protein

A coated pit and a coated vesicle formed during receptormediated endocytosis (TEMs)

Plasma membrane 0.25 m

Endocytosis
In pinocytosis, molecules are taken up when extracellular fluid is gulped into tiny vesicles also called constitutive endocytosis

Constitutive Endocytosis
can be imaged with fluorescent vital stains aminostyryl pyridinium molecules

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FM 1-43

nonfluorescent in aqueous environment rapid resonance across ethylene bonds stabilized in nonpolar environments such as phospholipid bilayers

FM 1-43

rapid resonance; weak fluorescence

stable resonance; strong fluorescence

FM 1-43
initially used to study events at the neuromuscular junction synaptic vesicle recycling many studies subsequently use for a variety of dynamic membrane events like endocytosis in mammals and amphibians (must read review paper on Bb) can be used in Tetrahymena

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


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 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)