Endo/exocytosis in Endo/exocytosis in the pollen tube the pollen tube

apex is apex is differentially differentially

regulated by Ca2+ regulated by Ca2+ and GTPasesand GTPasesPresented byPresented by

XIA,FanXIA,Fan

IntroductionIntroduction

Rapid extending cells: exocytosis , Rapid extending cells: exocytosis , endocytosis, and recyclingendocytosis, and recycling

[Ca2+] gradient[Ca2+] gradient GTP-binding proteins: GTP-binding proteins: o Rab: necessary for membrane traffic Rab: necessary for membrane traffic

between ER and Golgi.between ER and Golgi.o Rop: F-actin, [Ca2+]c gradientRop: F-actin, [Ca2+]c gradient

ResultResult

FM 1-43 dye is a valid marker for FM 1-43 dye is a valid marker for studying endo/exocytosisstudying endo/exocytosis

o FM 1-43 is highly fluorescent in a FM 1-43 is highly fluorescent in a membrane environment. Its membrane environment. Its permanent charge prevents the dye permanent charge prevents the dye from passively crossing the from passively crossing the membrane.membrane.

ResultResult

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

ResultResult

ResultResult Cytosolic calcium modulates endo/exocytosisCytosolic calcium modulates endo/exocytosiso The increase of [Ca2+]c upon release did not The increase of [Ca2+]c upon release did not

alter significantly the growth rate.alter significantly the growth rate.o But it affect the endo/exocytosis.But it affect the endo/exocytosis. GTPase activity lead to alternations in GTPase activity lead to alternations in

endo/exocytosisendo/exocytosiso The release of GTPrS in the cytoplasm led to The release of GTPrS in the cytoplasm led to

a significant increase in pollen tube growth a significant increase in pollen tube growth rate while the microinjection of GDPbS led to rate while the microinjection of GDPbS led to a decrease in growth rate.a decrease in growth rate.

o Photo activation of GTPrS led to a decrease Photo activation of GTPrS led to a decrease in FM 1-43 apical fluorescence.in FM 1-43 apical fluorescence.

ResultResult

ResultResult

Cross-talk between [Ca2+]c and Cross-talk between [Ca2+]c and GTPasesGTPases

Why [Ca2+]c decrease?

ResultResult Knock-down experimentKnock-down experiment

o The perturbation of polar growth can be accomplished by reducing the expression of Rop proteins.

o Provided that the growth is not totally disrupted, a [Ca2+]c gradient and, therefore, Ca2+ influx, can be maintained.

o The Rop inhibition results in a substantial decrease in the rate of FM 1-43 uptake, but not significant changes in the fluorescence pattern.

DiscussionDiscussion 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 Cell growth is not strictly dependent on a Ca2+-mediated

simulation of exocytosis. The exocytosis serves mainly to simulation of exocytosis. The exocytosis serves mainly to secrete cell wall component. secrete cell wall component. (The decrease of the (The decrease of the fluorescence is due to increase of secretion?)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 Fluorescence decrease is likely to due to the increase in

exocytosis.exocytosis.o GTPases play an active role in exocytosis by coupling the GTPases play an active role in exocytosis by coupling the

actin cytoskeleton to the sequential steps underlying actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.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 A large number of Rho family fit the model, and possible

candidates are Rops.candidates are Rops.o Rop protein is involved in endo/exocytosis and membrane Rop protein is involved in endo/exocytosis and membrane

retrieval, but may not be crucial for secretory vesicle retrieval, but may not be crucial for secretory vesicle targeting.targeting.

DiscussionDiscussion Rapid endocytosis in pollen tubes?Rapid endocytosis in pollen tubes?o It is a Ca2+-dependent process, coupled to exocytosis, that It is a Ca2+-dependent process, coupled to exocytosis, that

requires GTP hydrolysis and dynamin. Dynamin is a GTPase, requires GTP hydrolysis and dynamin. Dynamin is a GTPase, which require the hydrolysis of GTP to accomplish the which require the hydrolysis of GTP to accomplish the function. function.

[Ca2+]c and GTP control endo/exocytosis in a concerted but [Ca2+]c and GTP control endo/exocytosis in a concerted but differential waydifferential way

o Ca2+ plays a major role in the secretion of cell wall Ca2+ plays a major role in the secretion of cell wall components while ROP GTPases appear to play a key role in components while ROP GTPases appear to play a key role in fusion of docked vesicles and endocytosis.fusion of docked vesicles and endocytosis.

• Actin depolymerization is initially required to allow Actin depolymerization is initially required to allow membranes to dock.membranes to dock.

• Calcium only affects the excytosis process. Annexins is Calcium only affects the excytosis process. Annexins is needed to form the dock site. Relocation of annexins from the needed to form the dock site. Relocation of annexins from the cytosol to the membranes started when [Ca2+]c was elevated cytosol to the membranes started when [Ca2+]c was elevated to 300nM and reaching a maximum level at 800nM. to 300nM and reaching a maximum level at 800nM.

• The final membrane fusion process require the The final membrane fusion process require the reestablishment of an actin network.reestablishment of an actin network.

Endocytosis in PlantsEndocytosis in Plants

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