Effect of Overexpression of PSTPIP1 in Slug Formation and Phototaxis in Dictyostelium
Introduction
Dictyostelium discoideum are single-celled amoeboid protozoans currently being used in many
areas of biological research. They are ideal for research because they grow quickly and live happily at
room temperature. Furthermore, they form slugs, very primitive multicellular organisms, which allow for
comparison between single and multicellular stages in the same organism. Slug formation can be easily
stimulated by controlled conditions. While they are good models to use in general research, knowledge
gained from Dictyostelium cells can also be applied to human and animal cells. Dictyostelium cells are
more closely related structurally to animal cells than other model cells, like yeast (Williams, 2010).
Dictyostelium are used as a model for understanding cell migration. This area of research
is of particular interest because cell movement is such an important process seen throughout the
body, especially in regards to the immune system and metastasis of cancer cells. Inflammation,
for instance, is often the result of chemotaxis of white blood cells. Chemotaxis is the movement
of cells towards a “chemo-attractant.” The chemicals being released by a foreign body such as
bacteria or a virus. Similar to this, Dictyostelium slugs undergo phototaxis. This is specifically
cell migration toward a light source.
Current research has found that proteins in the Pombe Cdc15 homology (PCH) family are
involved in this process of cell migration. As these proteins are key in the cytoskeleton
formation, it would follow that they are also key components in making changes to that
formation to allow for movement (Lee et al. 2009). Determining the location of these proteins,
especially in relation to other proteins with known involvement with chemotaxis, such as WASP
(Wiskott - Aldrich syndrome protein). The process of chemotaxis is a series of steps in which
actin-filaments are polymerized, or elongated, creating a temporary “leading end” of the cell.
The cell is pulled forward as vesicles move cytoplasm from the back and the sides towards the
front of the cell. This has been supported by evidence of co-localization of PCH proteins around
the leading edge and sides of the cell around the vesicle formations during cell migration (Lee et
al. 2009). This study, in addition to one conducted by Heath and Insall, also portrayed that when
Dictyostelium lacking the genes coding for these PCH proteins (specifically forms of MEGAP)
were made, these cells were less motile. They did not move as quickly, nor did they move an
“organized” manner towards the target.
A study done by Heath and Insall (2008) also looked into how altering mgp1 and mgp2
affected slug formation of these cells. Slugs are a very primitive multicellular organisms that are
formed by Dictyostelium in adverse conditions. Slug formation can be stimulated by growing
cells on non-nutrient agar. In this type of adverse condition, cells will “communicate” via the
release cAMP and initiate aggregation (Williams, 2010). Therefore, because slug formation
depends heavily on cell migration, specifically chemotaxis, if cell migration is affected then it
stands that there might be an alterations seen in slugs as will. Slugs formed by cells lacking
mgp1- in the Heath and Insall study were shorter than those formed by wild type cells; however,
slugs formed by cells lacking the mgp2 were significantly longer (Heath and Insall, 2008).
Neither group of slugs moved as efficiently towards light during phototaxis as their wild type
counter parts (Heath and Insall, 2008). Although the exact process is still unknown, a significant
increase of vacuoles found in cells lacking either mgp1 or mgp2 support the reasoning that slugs
formed by these cells are abnormal due to a “mechanical” problem (Heath and Insall, 2008). As
discussed before, vacuoles play a large role in cell migration, and any defects are not only going
to impact the motility of the individual cell but also the motility and viability of the slug.
The study done by Heath and Insall (2008) also proposed that facilitating migration is the
expulsion of water out of vesicles formed by mgp (protein of PCH family); this expulsion, as
explained in the study, may help propel the cell forward during migration (Heath and Insall,
2008). The actual expulsion, as described by Heath and Insall 2008, is propagated by mgp. Mgp
surrounds the vacuoles near the membrane and expel the contents. Less mgp would mean less
force is being used to facilitate this expulsion, and the cell may be trying to compensate for the
lack of mgp by increasing the number of vacuoles working (Heath and Insall 2008).
PSTPIP1 (sometimes referred to as slp or pcha in Dictyostelium) is an interesting
member of the PCH family. Mutations of this protein have been linked to certain
autoinflammatory diseases (Hoing et al., 2013). It was found to affect vesicle formation in
Dictyostelium (Lee, 2009) and migration, but only knockdown studies were done and the
function of PSTPIP1 in the slugs was not addressed. Therefore, though the function of some of
the PCH family members have been studied, the role that the PSTPIP1 protein plays in cell
migration and slug phototaxis of Dictyostelium has not been fully explored. This research
investigates the localization of PSTPIP1 in the multicellular slug stage of the Dictyostelium life
cycle and determine the effect over-expression has on the formation and phototaxis of slugs.
Methods
Dictyostelium Growth
Although AX2 cells were used in preliminary stages of this experiment, KAX3 were the
wild-type Dictyostelium cells used in final analysis. Both were grown in suspension with HL5
media.
Electroporation
Electroporation was used to introduce DNA to the Dictyostelium cells. This process
involved applying electricity to the cell membranes in order to increase permeability and allow
for the uptake of the DNA plasmid. These pores close when the electric field is removed. The
DNA plasmid that used also contained resistance genes to G418. This drug was applied 24 hours
after electroporation, and any cells that were not producing these resistance genes (any cells that
did take up the DNA) were killed off. It took roughly one week to insure that surviving cells
were transformants. Once transformants are obtained, clonal lines will be made by transferring
cells to 96-well plate diluted to roughly 1 cell every 3 wells.
DNA used was a GPF-tagged PSTPIP1 and a control plasmid that only expresses GFP
(GFP-pLD1). DNA was generously provided by Douglas Robinson at Johns Hopkins University
School of Medicine.
Slug Formation
Slug formation was induced on 2% Agar (non-nutrient) plates. Cells were spun down in
centrifuge at 2500 rpm (700 rcf) and washed in a non-nutrient buffer (Sorenson’s buffer). Cells
were then recounted and suspended in enough Sorenson’s buffer to apply 2.5E7 cells suspended
in 50μL of buffer per plate. Plates were placed in a box with a slit for a single point of entry for
light. Each plate was marked where it aligned with the slit in order to analyze where slugs
traveled in relation to the placement of the light. Light will be placed so as to shine through the
slit and left overnight (~ 20 hours).
Analysis
Localization of GFP-PCHA was determined in multicellular form using fluorescent
microscopy. Number and size of slugs will be used to analyze slug formation. Analysis of
phototaxis was done by looking at the efficiency of slug movement; are slugs moving directly
towards the light or moving at odd angles? Imaging software ImageJ will be used for the
analysis of phototaxis. SPSS 18 will be used to analyze significance.
Results
AX2 cells overexpressing GFP-PSTPIP1 were successfully obtained using electroporation (Fig.
1) Furthermore, these cells could be induced to form slugs and PSTPIP1 overexpression could be
detected in both unicellular and multicellular stages (Fig. 1).
Figure 1. GFP-PSTPIP1 Transformants. Transformant cells were washed in a low-fluorescent media and pictures taken 4 hours
later (top). Transformant cells were plated on non-nutrient agar plates and placed in box with a slit for a point of entry for light.
Photos were taken 24 hours after plating (bottom). An Olympus IX-81 microscope with an Olympus XM-10 CCD camera and
CellSens software for acquisition and analysis was used for both individual cells (40X) and slugs (10X). Imaging soft-ware
ImageJ was used to create overlay images.
Literature Consulted
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Donghai, W. (2013). Inflammation in Mice Ectopically Expressing Human Pyogenic
Arthritis, Pyoderma Gangrenosum, and Acne (PAPA) Syndrome-associated PSTPIP1
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