Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells

Abstract

In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.

Figure 1.

Expression of full-length Syn6 and a truncated membrane-anchorless form (Syn6t), tagged with a c-myc epitope. (A) Western blotting with mAb 3D10 (against Syn6 cytosolic domain) of Syn6t-10, Syn6-2, or Syn6-21 cells either uninduced (-) or induced by the addition of 20 μM ponasterone (+) 24 h before cell lysis. (B) Western blot with a polyclonal anti-myc antibody in ponasterone induced Syn6t-10 cells incubated for the indicated times in 10 μg/ml cycloheximide to inhibit new protein synthesis; the identical samples were also probed with mAb 3D10 against Syn6. Note the presence of endogenous (“endog.”) Syn6 in A and B. (C) Immunofluorescence localization of endogenous TGN38 (primary mouse mAb, stained in green) and CPD (primary rabbit polyclonal antibody, stained in red) in Syn6t-10 cells in the absence and presence of inducer. An identical steady-state distribution was also observed upon induced expression of full-length Syn6 (our unpublished data).

Figure 2.

Extent of membrane association of inducibly expressed full-length Syn6 in Syn6-21 cells, and Syn6t in 6t-10 cells. (A) Homogenates were resolved into supernatant (S) and pellet (P) fractions as described in MATERIALS AND METHODS and analyzed by SDS-PAGE and Western blotting with antibodies against calnexin, myc tag, or GDI as indicated. (B) Immunofluorescence with anti-Syn6 mAb in uninduced (-) or induced (+) Syn6t-10 cells.

Figure 3.

Endocytic pathway phenotype associated with dominant-interfering Syn6t expression. (A) Lysosomal delivery/degradation of [³⁵S]SFV is inhibited by induced expression of Syn6t. The assay was performed as described in MATERIALS AND METHODS. After 2 h of warm up, the fraction of total radioactivity bound to uninduced Syn6-21 cells that became degraded and occurred as trichloroacetic acid-soluble counts in the medium was ∼35%; this was arbitrarily assigned a value of 1.0 and all other cells, conditions, and time points were compared with this value. The hatched lines indicate curves extrapolated to zero. (B) Recycling of ligand-bound transferrin receptors. After the cells indicated were loaded to steady state with ¹²⁵I-transferrin, the cells were washed and recycling of transferrin receptors to the cell surface was followed by release of ²⁵I-transferrin into the medium (and disappearance from cells).

Figure 5.

Progression of SFV through the endocytic pathway of INS cells. (A) Virus was prebound to the clones indicated at 4°C and the cells then washed thoroughly before warm up. At different times after warm up to 37°C, the cells were fixed, permeabilized, and immunostained using a mAb for the SFV E2 protein and a Cy3-conjugated secondary antibody (in red), and whole cell immunofluorescence captured with a charge-coupled device camera. (B) Samples were prepared as in A, but double labeled for SFV E2 (in red) and cation-independent mannose-phosphate receptors and a Cy2-conjugated secondary antibody (in green). The images were captured by confocal fluorescence.

Figure 4.

Continuous uptake of anti-Tac mAb in various INS cell clones transiently transfected to express a Tac-Lamp1 chimera. Transfected cells are indicated with arrows. Immunofluorescence distribution of cathepsin B-positive lysosomes is shown in green. The 2-h uptake of anti-Tac is detected by a Cy3-conjugated secondary antibody, shown in red. Regardless of the presence or absence of ponasterone, in cells not expressing Syn6t the Tac-Lamp1 chimera reaches cathepsin B-positive compartments. Note that in cells expressing Syn6t, the Tac-Lamp1 chimera shows delayed delivery to cathepsin B-positive compartments (bottom right).

Figure 6.

Biosynthesis of mature insulin secretory granules is not detectably affected by induced expression of Syn6t. (A) Syn 6t-10 cells either uninduced (-) or induced for 24 h with ponasterone (+) were pulse labeled, chased, and stimulated with secretagogue as described in the text. Proinsulin (Proins) and insulin (Ins) were recovered by immunoprecipitation with an anti-insulin antibody followed by SDS-PAGE and fluorography. (B) The cells as described below each panel were processed for electron microscopy as described in MATERIALS AND METHODS. A portion of the nucleus of the cell is oriented to the left of each image. The cells show relatively abundant secretory granules and mitochondria.

Figure 7.

Biogenesis of new insulin secretory granules in cells expressing full-length Syn6 or Syn6t. Multiple wells of Syn 6-21 cells (open symbols) or 6t-10 cells (closed symbols) were induced for 24 h with ponasterone pulse labeled for 30 min and then chased for 30-min blocks of time: 0–30, 30–60, 60–90, and 90–120 min. The endpoint of each chase collection period is shown along the x-axis. During each independent 30-min chase period, samples were examined under stimulated or unstimulated conditions as in Figure 6. At the conclusion of each period, the collected media and cell lysates were immunoprecipitated with anti-insulin and analyzed by SDS-PAGE and phosphorimaging. The band intensities for proinsulin, conversion intermediates, and insulin were quantitated in each sample. (A) Acquisition of stimulus competence of new secretory granules. Percentage of secretion during each time interval was calculated by the sum of proinsulin + conversion intermediates + insulin in the medium as a fraction total present in the cell lysate plus the medium collected during that time interval. The percentage of stimulus-dependent secretion, a measure of acquisition of stimulus competence, was calculated as the percentage of secretion under stimulated conditions minus the percentage of secretion under unstimulated conditions. The mean and value ranges in two identical experiments are shown (range was the same magnitude in both cell lines; shown for one set of cells only as the ranges between the cell lines overlap). (B) Processing of proinsulin to insulin within releasable secretory granules. From the stimulus-dependent secretion, the fraction comprised of proinsulin (circles), conversion intermediates (triangles), and insulin (squares) is shown as a function of time. The mean and range of values show the relative insulin production as indicated on the Figure. Note that by 2 h of chase, conversion to insulin in both sets of cells is indistinguishable.

Figure 8.

A minor lysosomal enzyme missorting defect is observed in INS cells upon induced expression of Syn6t. (A) Syn 6-21 cells (top) or Syn6t-1 or Syn6t-10 cells (bottom) either uninduced (-) or induced for 24 h with ponasterone (+) were pulse labeled and either lysed without chase (Pulse) or chased in the absence of secretagogues for 3 h (Chase). ProB and mature cathepsin B were recovered by immunoprecipitation with an anti-ProB antibody followed by SDS-PAGE and fluorography (right). In addition, lysates from the cells without chase were immunoprecipitated with anti-myc antibody to confirm induced expression of Syn6 or Syn6t (panels at left). The molecular weight markers were prestained and should be considered only approximations. (B) Syn6t-10 cells either uninduced (-) or induced for 24 h with ponasterone (+) were pulse labeled for 30 min and chased for the times indicated in the absence -) or presence (+) of secretagogue stimulation (Stim). The identical media and cell lysate samples were immunoprecipitated with anti-ProB antibody (top) and anti-insulin antibody (bottom). The positions of ProB, mature cathepsin B (mature B), proinsulin (Proins), and insulin (Ins) are shown.

Figure 9.

Transient expression and recycling of albumin-CPD and Tac-TGN38 chimeras in INS cells. Experiment : Syn 6-21 cells or Syn6t-10 cells were transfected with a plasmid encoding Alb-CPD and either uninduced (-) or induced for 24 h with ponasterone (+) before a 2-h continuous uptake with an anti-albumin polyclonal antibody (in red) as well as the immunofluorescent distribution of TGN38 by using a mAb (in green). In cells expressing Syn6t, transfected cells are marked with small arrows. Experiment : INS cells were transfected with Alb-CPD, full-length Syn6, or Syn6t-encoding plasmids, alone or in combination as indicated (double transfections used a 5:1 ratio of syntaxin DNA to Alb-CPD DNA). The cells were then labeled as in experiment . In cells expressing Syn6t, transfected cells are marked with small arrows. Experiment : Syn 6-21 cells or Syn6t-1 cells were transfected with a plasmid encoding Tac-TGN38 and either uninduced (-) or induced for 24 h with ponasterone (+) before a 2-h continuous uptake with a mAb anti-Tac (in red) as well as the immunofluorescent distribution by using a polyclonal anti-TGN38 lumenal domain (in green). In the latter two panels, the nuclei are also stained with 4,6-diamidino-2-phenylindole (in blue). Transfected cells are marked with arrows. Experiment : INS39 cells or Syn6t-10 cells were transfected, induced with ponasterone, and labeled as in experiment , except that the cells were examined by confocal fluorescence microscopy.

Figure 10.

Syn6t expression alters the distribution of Vti1b in INS cells. Control INS cells or Syn6t-10 cells were treated with ponasterone for 24 h, lysed, and the cell extracts loaded on sucrose velocity gradients. The numbered fractions run from top to bottom of the sucrose gradients. Each fraction was analyzed by SDS-PAGE and immunoblotting with the antibodies indicated. Small horizontal lines to the left of the gels indicate positions of prestained molecular mass markers that allowed for identification of the correct region. Clnx, calnexin; EEA1, early endosomal antigen 1; p38, synaptophysin.

Figure 11.

Vti1b association with Syn6t and Syn6 in INS cells. The cell lines indicated were immunoprecipitated with anti-myc mAb or anti-myc polyclonal antibody, which were then subjected to SDS-PAGE and immunoblotting. All three lower panels show immunoblotting with anti-Vti1b; note that from control INS39 cells no Vti1b can be coprecipitated with anti-myc (left and middle) although Vti1b can be coprecipitated when the cells are immunoprecipitated with anti-Syn6 (bottom right). The upper panels all show myc Western blotting with polyclonal anti-myc to confirm direct immunoprecipitation of the myc-tagged Syn6 full-length and Syn6t constructs as well as the presence of these bands in the original cell lysates (top right).