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 µm

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 µm

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