In complementary studies, we are investigating the role of CH domains in budding and pseudohyphal yeast cells by examining the phenotypes of mutants in yeast calponin and by identifying who and how yeast calponin interacts with other proteins. [Matsudaira, Goodman in collaboration with G. Fink]

Macrophage Cytoskeleton and Podosomes

Using IC-21 and P388D1 murine macrophages we investigate the formation and turnover of actin-rich adhesive bodies called podosomes. Podosomes are also present in osteoclasts and some transformed cells, these highly dynamic structures are found at the leading edge of the migrating cell and exhibit extraordinary morphology during their brief existence.

Murine IC-21 Macrophage
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A murine IC-21 macrophage recorded by phase contrast microscopy as it crawls across a glass surface. Images were taken at 30 second intervals.

Podosomes contain a multitude of proteins, many of which are also found in focal adhesions present in cells such as fibroblasts. Unlike the elongated focal adhesions found in fibroblasts, podosomes are punctate and highly dynamic. In our lab we have previously shown that fimbrin forms an interface between the microfilament cytoskeleton and the intermediate filament subunit, vimentin (see Correia et al. J Cell Biol. 146:831-42 1999) . Using a combination of biochemical and microscopy-based techniques, we are investigating the structural dynamics associated with podosome generation and turnover.

The vimentin intermediate filament network (red) extends into the lamella where tetramers colocalize with the actin-bundling protein, fimbrin (green). These fluorescent foci are specialized focal adhesions found in osteoclasts and macrophages called podosomes.

Podosomes contain fimbrin (green) and actin (red) and are found at the leading and retracting edges. In addition, these adhesion complexes show close association with microtubules (blue). This image was collected by confocal microscopy and deconvolved using Huygens2 then rendered in Imaris3 using a shadow algorithm.

Regulation of the yeast cytoskeleton by overlapping functions of fimbrin and Scp1p

Our goal is to understand how actin-binding proteins regulate dynamics and functions of the actin cytoskeleton. We are studying the functions of two yeast actin-binding proteins: fimbrin (Sac6p) and Scp1p. While fimbrin has been extensively studied in both lower and higher eukaryotes, Scp1p is a previously uncharacterized protein, highly conserved from yeast to mammals. We have found that Scp1p shares some functions with fimbrin. The cellular roles of the yeast fimbrin include morphogenesis, endocytosis, and polarized secretion. Like fimbrin, Scp1p localizes to cortical patches and stabilizes filamentous actin. Although loss of Scp1p function does not produce any discernable phenotype, deletion of SCP1 enhances many fimbrin mutant phenotypes. Moreover, increased dosage of SCP1 suppresses specific fimbrin phenotypes. We have both genetic and biochemical evidence for the role of Scp1p in regulation of stability and dynamics of the actin cytoskeleton. We have also found that Scp1p carries out an actin-binding independent function, which is partially redundant with fimbrin functions. Scp1p may serve as an adapter in endocytosis. Scp1p physically interacts with yeast epsins Ent1p and Ent2p, clathrin-binding proteins recently implicated in endocytosis by Wendland and colleagues. Thus, Scp1p may provide the long-sought molecular link between actin cytoskeleton and endocytic machinery.

GFP-SCP1 fluorescence localizes to polarized cortical patches.