HAP1Regulates Micro Filament-based Transport For Recruitment Of Secretory Granules To Cell Membrane In Pancreatic β Cells

Huntingtin-associated protein1(HAP1) is the first protein identified as an interacting partner of huntingtin, which is directly associated with Huntington’s disease. HAP1consists of two alternatively spliced isoforms, HAP1A and HAP1B, which differ only in their short C-terminal sequences(residues579-599in HAP1A and579-629in HAP1B). HAP1is a cytoplasmic protein enriched in neurons, the deletion of which in mice leads to neurodegeneration in the hypothalamus, feeding defects, retarded growth, and early postnatal death. HAP1binds to huntingtin in proportion to the number of glutamine repeats, indicating disruption of the normal cellular functions of HAP1may contribute to the onset and progression of Huntington’s disease.HAP1appears to play multiple roles in the cell. One well-supported role seems to be as an adaptor protein linking various vesicle cargos to microtubules. In particular, HAP1, especially HAP1A, is known to bind the microtubuleassociated motor proteins dynactin p150Glucd (p150) and kinesin light chain (KLC). Through these interactions, HAP1helps regulate microtubule-dependent vesicular trafficking essential for neuronal survival and function.HAP1is also expressed in endocrine cells, including pancreatic P cells. In previous work, we and our collaborators used the Cre-loxP system to generate mice in which HAP1expression was selectively depleted in pancreatic β cells. These mice showed impaired glucose tolerance and lower release of insulin in response to intraperitoneal glucose injection, suggesting that HAP1participates in insulin secretion by β cells. We further showed that, as in neurons, HAP1A binds p150and KLC, suggesting that HAP1participates in microtubule-dependent trafficking in endocrine cells as well as in neurons. Together, these results indicated that HAP1helps drive microtubule-based intracellular trafficking of insulin-containing granules in β cells. It raised the possibility that HAP1maybe involved in microfilament-based recruitment of insulin-containing granules to the cell surface. The microfilament-dependent motor protein myosin Va recruits secretory granules to the cortical area in β cells. Knocking down myosin Va expression using RNA interference significantly reduced glucose-stimulated insulin secretion, while expressing the dominant-negative globular tail domain of myosin Va led to50%fewer secretory granules docked at the plasma membrane.The present study aimed to probe whether HAP1associates with myosin Va and participates in the microfilament-based recruitment of insulin-containing granules to the cell surface in β cells.1. HAP1colocalizes with insulin, myosin Va and actin in β cells Immunostaining cultured INS-1cells for HAP1revealed a punctate distribution in the cytoplasm, including the processes. If HAP1is involved in the transport of insulin secretory granules along actin filaments, it should interact with insulin secretory granules and microfilament-based motor protein myosin Va. Using confocal laser scanning microscopy combined with immunofluorescence double-labeling to track HAP1and insulin simultaneously, we found that HAP1colocalized with insulin secretory granules in INS-1cells, especially in the cortical area. Repeating the double-labeling experiments to track HAP1and myosin Va showed partial colocalization. Double-immunofluorescence staining for myosin Va and actin or for HAP1and actin showed that the proteins in each pair co-localized with each other. The co-localized relationship between HAP1/insulin, HAP1/myosin Va and HAP1/actin indicates that HAP1may participates in the microfilament-based recruitment of insulin-containing granules by associates with insulin-containing granules and myosin Va.2. HAP1A comigrates with insulin-containing secretory granules in the peripheral area of β cells The alternatively spliced isoforms HAP1A and HAP1B have different C-terminal sequences. To gain direct evidence that one or both isoforms may help drive microfilament-dependent transport of insulin secretory granules, we transiently transfected INS-1cultures with phogrin-EGFP as a marker of insulin-containing granules in combination with either HAP1A-Dsred or pJred-HAP1B. HAP1A-Dsred showed a punctate distribution in the cytoplasm and colocalized with insulin-containing granules. We also observed by time-lapse confocal scanning microscopy that HAP1A moved with insulin-containing secretory granules in the peripheral area of β cells. In contrast to HAP1A, HAP1B showed no evidence of movement and remained diffusely distributed throughout the cytoplasm. These results suggest that the HAP1A may play a more important role than HAP1B in secretory granule movement in β cells.3. Silencing of HAP1inhibits high glucose induced recruitment of insulin-containing secretory granules to the cell surface If HAP1is involved in micro filament-dependent transport in the cortical area, altering HAPl expression levels should affect the distribution of intracellular insulin secretory granules in the vicinity of the cell membrane. To test this idea, HAP1siRNA was transiently transfected into INS-1cells with phogrin-EGFP as a marker of insulin-containing granules. Cells were stimulated for10min with20mM glucose to recruit insulin secretory granules to the cell surface.Laser scanning confocal microscopy (LSCM) was use to capture the images before and after glucose stimulaiton respectively. The ratio of EGFP fluorescence in the peripheral area of the cell (outermost10% of the inner cell area) to the total EGFP fluorescence within the entire cell area after glucose stimulation in the presence of HAP1siRNA or scrambled siRNA was measured. This ratio was significantly lower in HAP1siRNA-treated cells than in cells treated with scrambled siRNA. The result indicated that silencing of HAP1inhibited glucose-induced recruitment of secretory granules to the cell surface significantly.Total internal reflection fluorescence microscopy (TIRFM) showed that the number of secretory granules recruiting to the cell surface in HAP1siRNA-treated cells was significantly decreased compared with that of secretory granules in cells treated with scrambled siRNA.The results observed by LSCM and TIRFM showed that HAP1could regulate the recruitment of insulin containing secretory granules to the cell surface.4. Silencing of HAP1reduces the interaction between myosin Va and insulin-containing granules Using co-immunoprecipitation experiments, we showed that HAP1, myosin Va and phogrin interacted with each other. To probe further the possibility that HAP1acts an adaptor to connect myosin Va and insulin-containing granules, siRNA was used to knockdown HAP1expression level. After INS-1cells were stimulated with20mM glucose for10min, the association of myosin Va with phogrin was significantly reduced, which may explain the reduced distribution of the recruitment of secretory granules to the cell membrane observed by LSCM and TIRFM. These findings provided strong evidence that HAP1does act as an adaptor to link myosin Va and vesicles.5. The interaction between HAP1and myosin Va is increased by high glucose stimulation, which is involved with the dephosphorylation of HAP1A The extent of the interaction between HAP1and myosin Va visibly increased after glucose stimulation, which was observed by co-immunoprecipitation study. To gain more evidence of an interaction between HAP1and myosin Va, we replaced the598threonine(T598, the phosphorylated residue of HAPIA) with alanine (A), generated expression plasmids encoding full-length wild-type HAP1A and dephosphorylated mutant HAPIA (T598A), and transfected them into cells. Co-immunoprecipitation experiments showed that dephosphorylated HAPIA pulled down more myosin Va than wild-type HAP1A, which indicated that the increased interaction between HAP1and myosin Va after high glucose stimulation was related with the de phosphorylation of HAP1A.Conclusion HAP1could act as an adaptor to link myosin Va and insulin-containing granules, regulate microfilament-based transport of insulin containing granules in pancreatic β cells.