Cristina R. Artalejo

Quantal Size Is Dependent on Stimulation Frequency and Calcium Entry in Calf Chromaffin Cells

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.

Secretion: Dense-core vesicles can kiss-and-run too

New measurements show that the entire transmitter contents of a dense-core vesicle can be released within a second through a narrow fusion pore that opens transiently. With other results, this raises the possibility that some dense core vesicles may, like small synaptic vesicles, undergo immediate recycling.

A GAPDH Mutant Defective in Src-Dependent Tyrosine Phosphorylation Impedes Rab2-Mediated Events

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2‐mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2‐dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane‐binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose‐dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus‐G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C ι/λ to GAPDH Y41F, which reduces β‐coat protein association with the VTC and subsequent formation of Rab2‐mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.

Src-dependent aProtein Kinase C ι/λ (aPKCι/λ) Tyrosine Phosphorylation Is Required for aPKCι/λ Association with Rab2 and Glyceraldehyde-3-phosphate Dehydrogenase on Pre-Golgi Intermediates

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C ι/λ (aPKCι/λ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCι/λ and GAPDH bind directly to Rab2 and aPKCι/λ and GAPDH interact. Based on the reports demonstrating aPKCι-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCι/λ and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCι/λ on the VTC. To determine the consequence of aPKCι/λ tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCι/λ. Since Rab2 requires aPKCι/λ for the downstream recruitment of β-coat protein (β-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of β-COP. Like aPKCι/λ, the membranes contained a negligible amount of β-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCι/λ facilitates aPKCι/λ association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of β-COP and release of Rab2-mediated retrograde-directed vesicles.

Ageing changes the cellular basis of the “fight-or-flight” response in human adrenal chromaffin cells

Stress-induced increases in plasma epinephrine in man have been reported to decrease with age. To investigate the possible cellular basis for this decline we determined the characteristics of calcium currents and their relationship to catecholamine secretion in isolated human adrenal chromaffin (AC) cells. Cells derived from young individuals displayed prominent prepulse facilitation of L-type Ca channels but this property was absent in cells from older subjects. Robust quantal secretion in young cells as determined by amperometry was strongly coupled to the activation of these channels with an average delay of only approximately 3 msec. N- and P-type Ca channels also contributed to secretion but were more weakly coupled to catecholamine release sites. Cells from older subjects secreted much less efficiently and showed only weak coupling between Ca channels and secretion. These studies suggest that the magnitude and timing of adrenal secretion changes with age and that the facilitation Ca channel is key to rapid activation of the fight-or-flight response in young individuals.

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism.

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin‐1 dependent but clathrin‐independent and slow endocytosis (SE), dynamin‐2 and clathrin‐dependent. Various recombinant SH3 domains that interact with the proline‐rich domain of dynamin were introduced into single cells via the patch pipette. GST‐SH3 domains of amphiphysin‐1, intersectin‐IC, and endophilin‐I inhibited SE but had no effect on RE. Grb2‐SH3 (N‐terminal) or a mutant of amphiphysin‐1‐SH3 was inactive on either process. These data confirm that dynamin‐1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin‐2‐dependent SE.

Atypical Protein Kinase C Plays a Critical Role in Protein Transport from Pre-Golgi Intermediates

The small GTPase Rab2 requires atypical protein kinase C ι/λ (PKCι/λ) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702–712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCι/λ in the early secretory pathway. A peptide corresponding to the unique PKCι/λ pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCι/λ, β-COP, and p53/p58. Because Rab2, β-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCι/λ (W274K). Both the pseudosubstrate peptide and kinase-dead PKCι/λ in tandem with Rab2 caused sustained membrane association of PKCι/λ, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCι/λ was reversed by PKCι/λ wild type. These combined results indicate that PKCι/λ is essential for protein transport in the early secretory pathway and suggest that PKCι/λ kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro234 to Ser (Pro234Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro234Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro234Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively.

Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation

Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration.

Src-Dependent Apkcι/λ Tyrosine Phosphorylation Is Required For Apkcι/λ Association With Rab2 And Glyceraldehyde-3-Phosphate Dehydrogenase On Pre-Golgi Intermediates

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C ι/λ (aPKCι/λ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCι/λ and GAPDH bind directly to Rab2 and aPKCι/λ and GAPDH interact. Based on the reports demonstrating aPKCι-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCι/λ and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCι/λ on the VTC. To determine the consequence of aPKCι/λ tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCι/λ. Since Rab2 requires aPKCι/λ for the downstream recruitment of β-coat protein (β-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of β-COP. Like aPKCι/λ, the membranes contained a negligible amount of β-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCι/λ facilitates aPKCι/λ association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of β-COP and release of Rab2-mediated retrograde-directed vesicles.