TY - JOUR
T1 - SNAREs, HOPS and regulatory lipids control the dynamics of vacuolar actin during homotypic fusion in S. Cerevisiae
AU - Karunakaran, Surya
AU - Sasser, Terry
AU - Rajalekshmi, Sailasree
AU - Fratti, Rutilio A.
PY - 2012
Y1 - 2012
N2 - Homotypic vacuole fusion requires SNAREs, the Rab Ypt7p, the tethering complex HOPS, regulatory lipids and actin. In Saccharomyces cerevisiae, actin functions at two stages of vacuole fusion. Pre-existing actin filaments are depolymerized to allow docking and assembly of the vertex ring (a microdomain enriched in proteins and lipids that mediate fusion). Actin is then polymerized late in the pathway to aid fusion. Here, we report that the fusion machinery regulates the accumulation of actin at the vertex ring. Using Cy3-labeled yeast actin to track its dynamics, we found that its vertex enrichment was abolished when actin monomers were stabilized by latrunculin-B, independent of the extent of incorporation. By contrast, stabilization of filamentous actin with jasplakinolide markedly augmented actin vertex enrichment. Importantly, agents that inhibit SNAREs, Ypt7p and HOPS inhibited the vertex enrichment of actin, demonstrating that the cytoskeleton and the fusion machinery are interdependently regulated. Actin mobilization was also inhibited by ligating ergosterol and PtdIns(3)P, whereas the ligation or modification of PtdIns(4,5)P2 augmented the vertex enrichment of actin. The proteins and lipids that regulated actin mobilization to the vertex did not affect the total incorporation of Cy3-actin, indicating that actin mobilization and polymerization activities can be dissociated during membrane fusion.
AB - Homotypic vacuole fusion requires SNAREs, the Rab Ypt7p, the tethering complex HOPS, regulatory lipids and actin. In Saccharomyces cerevisiae, actin functions at two stages of vacuole fusion. Pre-existing actin filaments are depolymerized to allow docking and assembly of the vertex ring (a microdomain enriched in proteins and lipids that mediate fusion). Actin is then polymerized late in the pathway to aid fusion. Here, we report that the fusion machinery regulates the accumulation of actin at the vertex ring. Using Cy3-labeled yeast actin to track its dynamics, we found that its vertex enrichment was abolished when actin monomers were stabilized by latrunculin-B, independent of the extent of incorporation. By contrast, stabilization of filamentous actin with jasplakinolide markedly augmented actin vertex enrichment. Importantly, agents that inhibit SNAREs, Ypt7p and HOPS inhibited the vertex enrichment of actin, demonstrating that the cytoskeleton and the fusion machinery are interdependently regulated. Actin mobilization was also inhibited by ligating ergosterol and PtdIns(3)P, whereas the ligation or modification of PtdIns(4,5)P2 augmented the vertex enrichment of actin. The proteins and lipids that regulated actin mobilization to the vertex did not affect the total incorporation of Cy3-actin, indicating that actin mobilization and polymerization activities can be dissociated during membrane fusion.
KW - Actin dynamics
KW - Fusion
KW - HOPS
KW - Phosphoinositide
KW - Phospholipase C
KW - SNARE
UR - http://www.scopus.com/inward/record.url?scp=84863097996&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84863097996&partnerID=8YFLogxK
U2 - 10.1242/jcs.091900
DO - 10.1242/jcs.091900
M3 - Article
C2 - 22357954
AN - SCOPUS:84863097996
VL - 125
SP - 1683
EP - 1692
JO - The Quarterly journal of microscopical science
JF - The Quarterly journal of microscopical science
SN - 0021-9533
IS - 7
ER -