Figure 10. Myosin-VIIa clusters granules at cell corners.

(A–C) INS-1 832–13 cells were transfected with control siRNA duplexes or siRNA duplexes against the indicated proteins (Figure 9B, Figure 10—figure supplement 1) and were subjected to insulin secretion assays (A) as described in Figure 7B, or to insulin immunostaining (B, C) to examine the peripheral accumulation of granules as described in Figure 6C–E. (D) INS-1 832/13 cells were coimmunostained with anti-insulin antibody and either with anti-myosin-Va or -VIIa antibody. Bars, 10 μm. All quantitative data are means ± SD (n = 4). *p values calculated using two-tailed unpaired t-test are as follows: (A) 0.00029 (si Myosin-VIIa), 0.01168 (si Myosin-Va), 0.01881 (si RIM1), 0.00688 (si RIM2), 0.00045 (si Ca_v1.3) vs si Control, and (B) 6.2 × 10⁻⁵ (si Myosin-VIIa) vs si Control.

DOI: http://dx.doi.org/10.7554/eLife.26174.019

Figure 10—figure supplement 1. Silencing of myosin-Va, RIM, and Ca_v1.3 by siRNA.

INS-1 832/13 cells were transfected with control siRNA duplexes or siRNA duplexes against the indicated protein. The cell extracts were immunoblotted with the indicated antibody to evaluate its downregulation.

DOI: http://dx.doi.org/10.7554/eLife.26174.020

Figure 10—figure supplement 2. More images of INS-1 832–13 cells treated with siRNA.

INS-1 832/13 cells treated with siRNA against the indicated protein were immunostained with anti-insulin antibody, as shown in Figure 10B. Bars, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.26174.021

Figure 10—figure supplement 3. Localization of F-actin and its motor proteins.

(A) INS-1 832/13 cells expressing the C-terminal tail of HA-tagged myosin-VIIa (1550–2215 amino acids) or -Va (1444–1853 amino acids) were coimmunostained with anti-insulin and anti-HA antibodies. (B) INS-1 832/13 cells were costained with anti-insulin antibody and rhodamine-labeled phalloidin. Bars, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.26174.022