Endocytosis Cell Biology Lab Report

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A Sample Answer For the Assignment: Endocytosis Cell Biology Lab Report

Title:  Endocytosis Cell Biology Lab Report

Introduction:

This lab was conducted in two sessions to observe the properties of Dictyostelium

discoideum cells. The objectives of this experiment are to observe the part that actin plays in

actin-based processes and to identify the rates in which slime mold performs endocytosis and

exocytosis. The D. discoideum cell, or slime mold, was used as a model to observe the genes

which regulate the cytoskeleton and any processes requiring actin by using different mutants.

The cells are stained with DAPI (blue, showing where DNA is present) and rhodamine

conjugated phalloidin (red, showing where actin is present). In addition, the rates of endocytosis

and exocytosis can be compared using different mutants and evaluating their intake/outtake of

FITC-Dextran. In the instance of endocytosis, the concentration of FITC-Dextran should

increase in the slime mold cell, whereas during exocytosis, the concentration of FITC-Dextran

should decrease in the cell.

II. Materials and Methods:

In part 1, three lines of Dictyostelium discoideum are stained with DAPI and Rhoadmine

conjugated phalloidin. Differences are observed under a light microscope between the wild type

and mutant strains of the slime mold. Afterwards, each sample is treated with latrunculin, which

breaks up the actin into actin-g monomers. Again, the samples are observed for differences under

the microscope.

In part 2, six samples of slime mold are taken each for both endocytosis and exocytosis.

For the endocytosis, the cells are grown in a flask of HL5, then moved into a flask containing

HL5 and FITC-Dextran. The exocytosis is observed by growing the cells in HL5 and FITC-

Dextran first, then they are transferred into a HL5 containing flask before measuring. The

samples are obtained by lysing the cells and reading the relative fluorescence units.

III. Results: 10 micron images

The wildtype appears to have a mix of filopodia, pseudopodia, and lamellipodia actin structures.

The DAPI dye shows that the DNA is in an enclosed region in each structure, likely enclosed in

the nucleus

In the treated mutant 1, the slide has less compact areas with bright actin monomers, but also has

many dispersed areas stained blue, showing that there is no enclosed region with DNA.

Mutant 2 has small compact areas with DNA and compact areas with actin monomers. The actin

in Mutant 2, however, is less dense or more dilute than the wild type.

Average ENDO Line 0 20 40 60 80

WT 471 799 974 1344.5 1322

MT1 281 538.5 893.5 1,117 1140

MT2 810 1135.5 1344.5 1724 2168.5

0 10 20 30 40 50 60 70 80 90

0

500

1000

1500

2000

2500

Endocytosis

WT

MT1

MT2

Time (min)

RFU

Mutant 2 has an increased rate of endocytosis, whereas mutant 1 has a decreased rate of

endocytosis when compared to the wild type cell line