Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2+ and GTPases Presented by XIA,Fan

Introduction Rapid extending cells: exocytosis, endocytosis, and recycling Rapid extending cells: exocytosis, endocytosis, and recycling [Ca2+] gradient [Ca2+] gradient GTP-binding proteins: GTP-binding proteins: o Rab: necessary for membrane traffic between ER and Golgi. o Rop: F-actin, [Ca2+]c gradient

Result FM 1-43 dye is a valid marker for studying endo/exocytosis FM 1-43 dye is a valid marker for studying endo/exocytosis o FM 1-43 is highly fluorescent in a membrane environment. Its permanent charge prevents the dye from passively crossing the membrane.

Result FM 1-43 as a marker of polar growth FM 1-43 as a marker of polar growth

Result

Result Cytosolic calcium modulates endo/exocytosis Cytosolic calcium modulates endo/exocytosis o The increase of [Ca2+]c upon release did not alter significantly the growth rate. o But it affect the endo/exocytosis. GTPase activity lead to alternations in endo/exocytosis GTPase activity lead to alternations in endo/exocytosis o The release of GTPrS in the cytoplasm led to a significant increase in pollen tube growth rate while the microinjection of GDPbS led to a decrease in growth rate. o Photo activation of GTPrS led to a decrease in FM apical fluorescence.

Result

Result Cross-talk between [Ca2+]c and GTPases Cross-talk between [Ca2+]c and GTPases Why [Ca2+]c decrease?

Result Knock-down experiment Knock-down experiment oThe perturbation of polar growth can be accomplished by reducing the expression of Rop proteins. oProvided that the growth is not totally disrupted, a [Ca2+]c gradient and, therefore, Ca2+ influx, can be maintained. oThe Rop inhibition results in a substantial decrease in the rate of FM 1-43 uptake, but not significant changes in the fluorescence pattern.

Discussion Cytosolic calcium controls secretion of cell wall? Cytosolic calcium controls secretion of cell wall? o Cell growth is not strictly dependent on a Ca2+-mediated simulation of exocytosis. The exocytosis serves mainly to secrete cell wall component. (The decrease of the fluorescence is due to increase of secretion?) GTPases control growth and membrane retrieval? GTPases control growth and membrane retrieval? o Fluorescence decrease is likely to due to the increase in exocytosis. o GTPases play an active role in exocytosis by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis. A ROP protein affects endo/exocytosis and apical growth? A ROP protein affects endo/exocytosis and apical growth? o A large number of Rho family fit the model, and possible candidates are Rops. o Rop protein is involved in endo/exocytosis and membrane retrieval, but may not be crucial for secretory vesicle targeting.

Discussion Rapid endocytosis in pollen tubes? Rapid endocytosis in pollen tubes? o It is a Ca2+-dependent process, coupled to exocytosis, that requires GTP hydrolysis and dynamin. Dynamin is a GTPase, which require the hydrolysis of GTP to accomplish the function. [Ca2+]c and GTP control endo/exocytosis in a concerted but differential way [Ca2+]c and GTP control endo/exocytosis in a concerted but differential way o Ca2+ plays a major role in the secretion of cell wall components while ROP GTPases appear to play a key role in fusion of docked vesicles and endocytosis. Actin depolymerization is initially required to allow membranes to dock. Actin depolymerization is initially required to allow membranes to dock. Calcium only affects the excytosis process. Annexins is needed to form the dock site. Relocation of annexins from the cytosol to the membranes started when [Ca2+]c was elevated to 300nM and reaching a maximum level at 800nM. Calcium only affects the excytosis process. Annexins is needed to form the dock site. Relocation of annexins from the cytosol to the membranes started when [Ca2+]c was elevated to 300nM and reaching a maximum level at 800nM. The final membrane fusion process require the reestablishment of an actin network. The final membrane fusion process require the reestablishment of an actin network.

Endocytosis in Plants

(GTPase activation --- F-actin assembly, vesicle trafficking --- Ca2+ channel activated, F-actin disassembly and docking --- wall material depositing, absorbing Ca2+ influx at the wall ---F-actin reassembly and membrane fusion (effect on the channel, GTPase distribution?)--- endocytosis, membrane retrieval---GTPase inactivation )