RESUMO
The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD(-/-) ) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.
Assuntos
Células Cromafins , Vesículas Transportadoras/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Western Blotting , Pesquisas com Embriões , Camundongos , Camundongos Knockout , Biogênese de Organelas , Isoformas de ProteínasRESUMO
Synaptotagmin-1, the canonical isoform of the synaptotagmin family, is a Ca(2+) sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca(2+) sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca(2+) affinity and slower binding kinetics than synaptotagmin-1, matching the proposed properties for the second, slower Ca(2+) sensor. Here, we examined Ca(2+)-triggered exocytosis in chromaffin cells from KO mice lacking synaptotagmin-7, and from knockin mice containing normal levels of a mutant synaptotagmin-7 whose C(2)B domain does not bind Ca(2+). In both types of mutant chromaffin cells, Ca(2+)-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both synaptotagmin-1 and -7, only a very slow release component, accounting for approximately 30% of WT exocytosis, persisted. These data establish synaptotagmin-7 as a major Ca(2+) sensor for exocytosis in chromaffin cells, which, together with synaptotagmin-1, mediates almost all of the Ca(2+) triggering of exocytosis in these cells, a surprising result, considering the lack of a role of synaptotagmin-7 in synaptic vesicle exocytosis.
Assuntos
Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/metabolismo , Cálcio/metabolismo , Células Cromafins/metabolismo , Exocitose , Sinaptotagmina I/metabolismo , Sinaptotagminas/metabolismo , Animais , Células Cromafins/citologia , Deleção de Genes , Cinética , Potenciais da Membrana , Camundongos , Camundongos Knockout , Estrutura Terciária de Proteína , Sinaptotagminas/químicaRESUMO
The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Células Cromafins/metabolismo , Depsipeptídeos , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte/genética , Bovinos , Células Cromafins/ultraestrutura , Exocitose/efeitos dos fármacos , Exocitose/genética , Microscopia Eletrônica , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Células PC12 , Peptídeos Cíclicos/farmacologia , Ratos , Vesículas Secretórias/ultraestrutura , Tiazóis/farmacologia , Tiazolidinas , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPAssuntos
Exocitose , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Proteína rab3A de Ligação ao GTP/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Catecolaminas/metabolismo , Eletrofisiologia , Cinética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Células PC12 , Ratos , Serotonina/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Serotonina , TransfecçãoRESUMO
Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.
Assuntos
Proteínas de Transporte/biossíntese , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Proteínas do Tecido Nervoso , Neurotransmissores/metabolismo , Feocromocitoma/metabolismo , Simportadores/biossíntese , Inibidores da Captação Adrenérgica/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Dopamina/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Neurotransmissores/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Células PC12/efeitos dos fármacos , Ratos , Reserpina/farmacologia , Vesículas Secretórias/metabolismo , Serotonina/metabolismo , Serotonina/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Serotonina , Simportadores/genética , TransfecçãoRESUMO
Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice.