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

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

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

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

4. Result • FM 1-43 as a marker of polar growth

5. Result

6. Result • Cytosolic calcium modulates endo/exocytosis • The increase of [Ca2+]c upon release did not alter significantly the growth rate. • But it affect the endo/exocytosis. • GTPase activity lead to alternations in endo/exocytosis • 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. • Photo activation of GTPrS led to a decrease in FM 1-43 apical fluorescence.

7. Result

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

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

10. Discussion • Cytosolic calcium controls secretion of cell wall? • 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? • Fluorescence decrease is likely to due to the increase in exocytosis. • 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 large number of Rho family fit the model, and possible candidates are Rops. • Rop protein is involved in endo/exocytosis and membrane retrieval, but may not be crucial for secretory vesicle targeting.

11. Discussion • Rapid endocytosis in pollen tubes? • 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+ 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. • 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.

12. Endocytosis in Plants

13. (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 )