The membrane actin cytoskeleton undergoes a
carefully coordinated series of changes in organization that
direct the morphological development of a yeast cell. To
understand how the organization of the cytoskeleton changes in
response to a variety of signals, we have biochemically
identified several yeast proteins, including cofilin, fimbrin and
Abp1p, that bind to actin filaments and regulate the assembly
and/or organization of actin filaments. We have genetically
tested the roles of these proteins in the living cell, and have
used selections and screens to identify the genes that regulate
these proteins. As a result of this work, and the work in several
other laboratories, a large number of genes that control cellular
morphogenesis in budding yeast has been identified. The challenge
now is determine how the products of a large number of genes
cooperate to control the development of cell polarity and cell
shape.
To this end, we have established a
permeabilized cell model for polarized actin assembly. By gently
permeabilizing yeast under conditions that preserve the
arrangement of intracellular constituents, we have established an
in vitro assay that allows the assembly of exogenously
added fluorescent actin into the precise regions of the cell that
incorporate actin in vivo. This assembly is dependent on
the functions of Sla1p and Sla2p, two gene products that function
in the in vivo incorporation of actin at the cell cortex.
Moreover, we have obtained evidence for the requirement of GTP-binding proteins for the actin nucleation activity. Finally,
the assembly of actin in permeabilized yeast cells can be
modulated by the cell cycle control cyclin-dependent kinase/cyclin complex in a manner that mimics the in vivo
control. We are using the permeabilized cell assay to elucidate
the full regulatory pathways that control actin assembly in
living cells. Since actin and the proteins that bind to actin are
highly conserved, the results that we obtain in yeast are likely
to be applicable to more complex eukaryotes as well.
Elucidation of the molecular mechanisms
used to regulate actin assembly will require a detailed knowledge
of how actin subunits assemble into long polymers. Recently,
atomic models for the actin monomer and actin filament have been
developed. These advances provide a unique opportunity to
determine the mechanisms of actin mediated processes at the level
of atomic interactions. We are performing a structure-function
analysis of actin by mutating specific residues and assaying the
effects of these mutations on actin assembly in vitro and in
vivo. We are developing novel genetic and biochemical
approaches to mapping the binding sites of actin-binding proteins
on the actin filament, and to relating specific aspects of actin
structure to actin function in the living cell. Finally, genetic,
biochemical and structural studies of the low molecular weight
(16 kD) actin filament severing protein cofilin are being
performed to determine the molecular mechanism of filament
severing and to elucidate the regulatory pathways that control
the activity of this protein.
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