Trafficking Of Secretory Granules Research Articles

Oncogenic Ras requires activation for pathological levels of activity

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA Background: Previous studies have shown that the small G-protein Rab27A regulates the intracellular trafficking of secretory granules in various cell types, including islet beta cells. However, its role in mouse pancreatic acini is poorly understood. We utilized ashen mice, which lack Rab27A to investigate the function of Rab27A in pancreatic acinar cells. Aim: To investigate the effects of Rab27A deficiency on pancreatic acinar cell physiology and function. Methods: Isolated pancreatic acini were prepared from ashen or wildtype (WT) mouse pancreases by collagenase digestion and CCKor carbachol-induced amylase secretion was measured. Expression of Rab27A, Rab27B, Rab3D and digestive enzymes were measured by western blot. GTP-bound states of Rab27B and Rab3D in ashen or WT mouse acini were assessed by GST-pulldown assay. Localization of Rab27B and Rab3D in ashen and WT pancreases was shown by immunofluorescence. Results: Expression of Rab27A was detected in isolated WT mouse pancreatic acinar cells by western blot and RT-PCR. Expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. The GTP-bound states of Rab27B and Rab3D in ashen and WT mouse acini also remained constant. Acini from ashenmice showed lower basal level of amylase release but no obvious change in the entire dose response curves of CCKor carbachol-stimulated amylase release. Localization of Rab27B, Rab3D and amylase by immunohistochemistry was not obviously changed in acinar cells from both mice. Localization of Rab27A could not be determined using available antibody. Conclusions: Rab27A is present in mouse pancreatic acinar cells but not involved in the final secretory steps of zymogen granule secretion.

Identifying the function of small G-protein Rab27A in the secretion in mouse pancreatic acinar cells

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA Background: Previous studies have shown that the small G-protein Rab27A regulates the intracellular trafficking of secretory granules in various cell types, including islet beta cells. However, its role in mouse pancreatic acini is poorly understood. We utilized ashen mice, which lack Rab27A to investigate the function of Rab27A in pancreatic acinar cells. Aim: To investigate the effects of Rab27A deficiency on pancreatic acinar cell physiology and function. Methods: Isolated pancreatic acini were prepared from ashen or wildtype (WT) mouse pancreases by collagenase digestion and CCKor carbachol-induced amylase secretion was measured. Expression of Rab27A, Rab27B, Rab3D and digestive enzymes were measured by western blot. GTP-bound states of Rab27B and Rab3D in ashen or WT mouse acini were assessed by GST-pulldown assay. Localization of Rab27B and Rab3D in ashen and WT pancreases was shown by immunofluorescence. Results: Expression of Rab27A was detected in isolated WT mouse pancreatic acinar cells by western blot and RT-PCR. Expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. The GTP-bound states of Rab27B and Rab3D in ashen and WT mouse acini also remained constant. Acini from ashenmice showed lower basal level of amylase release but no obvious change in the entire dose response curves of CCKor carbachol-stimulated amylase release. Localization of Rab27B, Rab3D and amylase by immunohistochemistry was not obviously changed in acinar cells from both mice. Localization of Rab27A could not be determined using available antibody. Conclusions: Rab27A is present in mouse pancreatic acinar cells but not involved in the final secretory steps of zymogen granule secretion.

Myosin Vc Is a Molecular Motor That Functions in Secretory Granule Trafficking

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (approximately 30 nm/s) and microtubule independently, whereas tubules move rapidly (approximately 440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.

X‐ray structure of Danio rerio secretagogin: A hexa‐EF‐hand calcium sensor

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF-hand proteins represent a superfamily of calcium-binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa-EF-hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X-ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium-free form at 2.1-A resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF-hand motifs. The domains are arranged into a V-shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium-loaded calbindin D(28K) revealed a striking difference in the spatial arrangement of their domains, which involves approximately 180 degrees rotation of the first globular domain with respect to the module formed by the remaining domains.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

The role of myosin Va in secretory granule trafficking and exocytosis

It emerges that myosin Va plays multiple roles in the trafficking of SGs (secretory granules). In addition to a function in the capture and transport of newly formed SGs in the F-actin-rich cortex, myosin Va is implicated in late transport events of these organelles, which precede their exocytosis. Consistent with these roles, interactions of myosin Va with an array of well-known proteins involved in regulated protein secretion have been documented.

Stabilization of the receptor protein tyrosine phosphatase-like protein ICA512 in GH4C1 cells upon treatment with estradiol, insulin, and epidermal growth factor.

Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60-70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 +/- 0.35- and 1.6 +/- 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512's function and vice versa.