Live cell imaging of mitochondrial movement along actin cables in budding yeast

BACKGROUND: Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.     

Yeast formins Bni1 and Bnr1 utilize different modes of cortical interaction during the assembly of actin cables

The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.     

A dynamic formin-dependent deep F-actin network in axons

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.     

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.     

Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p-green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.     

Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division.

BACKGROUND: Both symmetric and asymmetric cell divisions are required for the generation of appropriate cell lineages during development. Wild-type Schizosaccharomyces pombe cells divide in a symmetric fashion to produce two similar rod-shaped daughter cells. Formins are proteins with conserved roles in cell polarity, cytokinesis, and the regulation of actin and microtubule cytoskeletons. RESULTS: Here, we identify and characterize a new S. pombe formin, for3p. for3 Delta mutant cells divide in an asymmetric manner; a mother cell divides medially to produce one daughter cell that develops into a monopolar cell and one daughter that develops into a bipolar cell. Both daughter cells recapitulate similar asymmetric lineages themselves. Inheritance of the bipolar pattern correlates with inheritance of the recent birth scar, not with asymmetry in the spindle pole bodies. for3 Delta mutants lack interphase actin cables and have delocalized actin patch and myo52p (type V myosin) distributions. for3 Delta cells have normal microtubule dynamics and cortical interactions but have defects in microtubule organization and increased numbers of microtubule bundles. for3p-GFP is localized at both cell tips in an actin-dependent manner and at the cell division site. CONCLUSIONS: for3p is a cell polarity factor required for interphase actin cable formation and microtubule organization. The for3 Delta phenotype suggests that cells are able to grow in a polarized manner even in the absence of functional actin cables and polarized distribution of actin patches. for3p and possibly actin cables are part of a regulatory network that ensures that cell divisions are symmetric.     

Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe

Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.     

A Highlights from MBoC Selection: Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803–2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.     

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.     

FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.     

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton- plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.     

Movement of cortical actin patches in yeast

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.     

New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.     

Ventral stress fibers induce plasma membrane deformation in human fibroblasts

Interactions between the actin cytoskeleton and the plasma membrane are important in many eukaryotic cellular processes. During these processes, actin structures deform the cell membrane outward by applying forces parallel to the fiber’s major axis (as in migration) or they deform the membrane inward by applying forces perpendicular to the fiber’s major axis (as in the contractile ring during cytokinesis). Here we describe a novel actin–membrane interaction in human dermal myofibroblasts. When labeled with a cytosolic fluorophore, the myofibroblasts displayed prominent fluorescent structures on the ventral side of the cell. These structures are present in the cell membrane and colocalize with ventral actin stress fibers, suggesting that the stress fibers bend the membrane to form a “cytosolic pocket” that the fluorophores diffuse into, creating the observed structures. The existence of this pocket was confirmed by transmission electron microscopy. While dissolving the stress fibers, inhibiting fiber protein binding, or inhibiting myosin II binding of actin removed the observed pockets, modulating cellular contractility did not remove them. Taken together, our results illustrate a novel actin–membrane bending topology where the membrane is deformed outward rather than being pinched inward, resembling the topological inverse of the contractile ring found in cytokinesis.     

Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method

Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocytic patches in fission yeast to increase the quality of the data extracted from quantitative microscopy movies. We present a universal method to achieve “temporal superresolution” by aligning temporal data sets with higher temporal resolution than the measurement intervals. These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP. We show that the time course of actin assembly and disassembly varies <600 ms between patches. Actin polymerizes during vesicle formation, but we show that polymerization does not participate in vesicle movement other than to limit the complex diffusive motions of newly formed endocytic vesicles, which move faster as the surrounding actin meshwork decreases in size over time. Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.     

The ARP2/3 complex,acting cooperatively with Class I formins,modulates penetration resistance in Arabidopsis against powdery mildew invasion

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant–microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant–pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC–ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC–ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

ARP2/3 complex, acting cooperatively with Class I formins, modulates actin patch formation beneath fungal penetration sites, contributing to the penetration resistance of Arabidopsis against powdery mildew invasion.     

Behavior of mitochondria, microtubules, and actin in the triangular yeastTrigonopsis variabilis

Summary Dimorphic yeastTrigonopsis variabilis is a unique species that can form either an ellipsoidal or a triangular cell depending upon nutritional conditions. This fluorescence microscopic study was intended to correlate morphological changes of mitochondria in the triangular cells with the distribution of the cytoskeleton. In addition, unique features in the behavior of the cytoskeleton were also examined during triangular cell formation. In log-phase cells stained with 4,6-diamidino-2-phenylindole, mitochondrial nucleoids appeared as a string of beads throughout the vegetative growth. The profile of mitochondria stained by 3,3-dihexyloxacarbocyanine iodide showed a network corresponding to the fluorescence images of mitochondrial nucleoids in both mother and daughter cells. Cell-cycle-dependent fragmentation of mitochondria was not discerned. As the culture reached stationary phase, a network of mitochondria gradually changed to form unique rings that were located near the angles of triangular cells. When examined by immunofluorescence microscopy with anti-tubulin antibody, microtubules were found to be well developed along the sides of cells in the cytoplasm ofT. variabilis interphase cells. Although distributions of microtubules and mitochondria are different during cell cycle as a whole, cytoplasmic microtubules frequently extended along a part of the mitochondria in budded cells, suggesting correlation of microtubules and mitochondria. Rhodamine-phalloidin staining revealed both actin patches and cables. Actin cables elongated from mother cells into the buds and showed close proximity to mitochondria, although complete overlapping of both structures was rare. Moreover, actin patches localized on the mitochondrial network at a frequency of 65%. These results suggested that actin cables and patches, as well as microtubules, participated in the distribution of mitochondria. The localization of actin patches separated towards opposite ends at a bud tip when the bud grew to medium size. The unique localization of actin patches is responsible for bi-directional growth of the bud, forming triangular cells.     

Sro9, a Multicopy Suppressor of the Bud Growth Defect in the Saccharomyces Cerevisiae Rho3-Deficient Cells, Shows Strong Genetic Interactions with Tropomyosin Genes, Suggesting Its Role in Organization of the Actin Cytoskeleton

RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth. SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3δ mutation. An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein). Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99. However, sro9δ was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9δ tpm1δ cells did not distribute cortical actin patches properly and lysed. We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1δ sro9δ double mutant. Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth. Overexpression of SRO9 suppressed the growth defect in tpm1δ tpm2δ cells, disappearance of cables of actin filaments in both rho3δ cells and tpm1δ cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function. Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic. These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.     

Visualization of receptor-mediated endocytosis in yeast

We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.     

Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis

The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process.