Movement of cortical actin patches in yeast.

Abstract

Abstract. In yeast, actin forms patches associated with the plasma membrane. Patch distribution correlates with polarized growth during the cell cycle and in response to external stimuli. Using green fluorescent protein fused to capping protein to image actin patches in living cells, we find that patches move rapidly and over long distances. Even patches in clusters, such as at the incipient bud site, show movement. Patches move independently of one another and generally over small distances in a local area, but they can also move larger distances, including through the mother-bud neck. Changes in patch polarization occur quickly through the cell cycle. These observations provide important new parameters for a molecular analysis of the regulation and function of actin.I r~ yeast, actin functions in polarized secretion and growth (6, 37). Actin filaments in yeast are found in cortical patches associated with invaginations of the plasma membrane and in cables running through the cytoplasm (26). The polarization of patches and the orientation of cables correlates with polarized cell growth (1). The distribution of both patches and cables changes dramatically over time during the cell cycle and in response to external stimuli (10). Cell cycle regulators control the redistribution processes (20), and several actin-binding proteins are necessary for patch polarization and cable formation (6, 37).Actin is essential for viability (31). Mutants lacking patches have never been observed, but mutants without cables are viable. Also, mutations in genes encoding other protein components of patches are lethal (2, 25). Therefore, patches may be the location of the essential function of actin. Temperature shift experiments indicate that actin is important for secretion (27) and endocytosis (19). Together, these observations suggest that the location of patch polarization specifies the location of polarized growth and cell wall remodelling.Therefore, we wish to understand how the location of cortical actin patches in a living yeast cell is controlled. In particular, we want to determine whether patches change their distribution by moving about or by disassembly at one place with re-assembly elsewhere. Also, we want to understand how rapidly the patches change between polarized and depolarized states. These pieces of information An analysis of actin patch dynamics is now possible in yeast with the advent of green fluorescent protein (GFP) t technology (33). We constructed a GFP derivative of the 13 subunit of capping protein (Cap2p) because capping protein is found only at cortical actin patches (3). GFP-Cap2p localized to patches and was able to function like Cap2p, rescuing a null mutant.We found that the distribution of patches changed on a time scale of seconds, much faster than landmark events through the cell cycle. Patches moved, at rapid rates and over long distances, even through the mother/bud neck. Even polarized patches, found in clusters at incipient bud sites, in the bud and at cell division sites, showed movement. A...

Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?

“…Among them, six showed a partial colocalization with actin bodies (Arc15p, Arc18p, Arc35p, Arp2p, Crn1p, and Srv2p), and five others (Abp1p, Abp140p, Cap1p, Cap2p, and Sac6p) strictly colocalized with actin bodies. Abp1p and Cap1p/ Cap2p are ABPs detected only in actin patches (Drubin et al, 1988;Amatruda and Cooper, 1992;Doyle and Botstein, 1996;Waddle et al, 1996), Abp140p is a protein preferentially found in actin cables (Asakura et al, 1998;Yang and Pon, 2002), and Sac6p has been detected in both actin cables and patches (Drubin et al, 1988;Doyle and Botstein, 1996). To confirm those results and increase when necessary the GFP signal in quiescent cells, 3xGFP fusion proteins were constructed and shown to be functional (see Materials and Methods).…”

“…Within these structures, protein exchange is very dynamic (Yang and Pon, 2002;Kaksonen et al, 2003), and the actin turnover in the filaments that form actin cables and patches is extremely rapid. Indeed, actin patches are very short-lived structures, the half-life of which is estimated to be 20 -40 s (Waddle et al, 1996). Consistently, in living cells, actin filaments rapidly disappear upon treatment with latruculin A (Lat-A), a drug that prevents G-actin polymerization (Ayscough et al, 1997).…”

Green fluorescent protein: applications in cell biology

“…One component of them, the capping protein (Cap2p), was fused to GFP and used as a reporter to monitor patch movement in living cells. Videomicroscopy revealed that changes in patch locations are due to movement of the patches and not to their disassembly and reassembly elsewhere [19].…”

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

“…Green fluorescent protein (GFP)-expressing cells were processed as described above with the exception that the spheroplasts were incubated for only 10 min at 37°C. Living spheroplasts were harvested by centrifugation and placed on microscope slide on a pad of 2% (wt/vol) agarose in PBS medium as described previously (Waddle et al, 1996). Cells were imaged using an Olympus AX70 Provis microscope equipped with a CoolSNAP HQ CCD camera controlled by IPlab Spectrum software (Scanalytics, Fairfax, VA).…”

“…One intriguing aspect of cortical actin patches is that they are highly motile (Doyle and Botstein, 1996;Waddle et al, 1996). The importance of actin patch motility in vivo is not yet understood, but there is evidence that motility may be initiated by Arp2/3 complex-nucleated actin polymerization (Winter et al, 1997;Kaksonen et al, 2003).…”