Student Theses and Dissertations

Date of Award

2011

Document Type

Thesis

RU Laboratory

Nurse Laboratory

Keywords

cell growth zone size, cell width, Cdc42, fission yeast, GTPases, spheroplasts

Abstract

The fission yeast Schizosaccharomyces pombe is a rod--‐shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it a good system to study the control of cellular dimensions. Here I used the width of the cell as a model for the control of growth zone size. To identify genes that influence cell size I carried out a near--‐genome--‐widescreen for mutants wider than wild--‐type cells. I found eleven deletion mutants that were wider; seven of the deleted genes are implicated in the control of the small GTPase Cdc42. Further analyses showed two different pathways are involved: one is defined by the Cdc42 guanine--‐nucleotide exchange factor (GEF) Scd1 and the second by the GTPase activating protein (GAP) Rga4. Deletions of rga4 and scd1 had additive effects on cell width, and the proteins localized independently of one another, with Rga4 located at the cell sides and Scd1 at the cell tips. Delocalization and ectopic retargeting experiments showed that these localizations are crucial for Rga4’s and Scd1’s roles in determining cell width. I propose that the GAP Rga4 and the GEF Scd1 establish a gradient of activated Cdc42 at the cellular tip, and it is this gradient that determines cell growth zone size and thus cell width. Cell wall removal generates round spheroplasts that can recover into normal rod--‐shaped cells, allowing the study of how a growth zone of a particular shape and dimensions is formed. Spheroplasts initially have a disorganized cytoskeleton and depolarized growth zone proteins. During recovery, these components repolarize and form one, or in some cases two, new growth zones from the rounded spheroplast body. Regenerated new growth zones have the same width as wild--‐type rod--‐shaped cells, and their width is controlled by the same genes that determine the width of normal exponentially growing cells. Scd2, a scaffold protein that is normally localized to the growth zone, forms a polarized patch in the rounded spheroplast before cell shape becomes polarized, showing that a growth zone protein can self--‐ organize independent of cell shape. Rga4 is initially randomly distributed along the round spheroplast membrane but is excluded from the de novo growth zone as growth begins. A stable patch of Scd2 forms before Rga4 is reorganized, suggesting that growth polarization may precede Rga4 exclusion. These results provide evidence that a cell’s growth zone can form de novo with the correct dimensions and that the cell’s rod shape is a cell--‐intrinsic, genetically controlled trait that does not depend on cell wall history. These studies show that the spatial regulation of Cdc42 is a major determinant of cell width in exponentially growing cells, and influences the width of a growth zone formed do novo. Cdc42 is an essential regulator of growth that is conserved throughout eukaryotes, and so this paradigm of spatial regulation may be broadly applicable.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/441

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