Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release.

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion.

Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca2+-dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.

Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids.

Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time-sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein-only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein-recruiting/protein-releasing granular depots.

Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

A convenient fluorimetry-based degranulation assay using RBL-2H3 cells.

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as ß-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of ß-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation.

VAMP7 knockdown in secretory granules impairs CCL2 secretion in mast cells.

Mast cells (MCs) possess numerous potent inflammatory mediators and undergo differential regulation in response to antigen (Ag) stimulation. Among the regulatory systems governing secretory responses, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in facilitating granule-plasma membrane fusion and subsequent secretion. Our previous investigation documented the involvement of vesicle-associated membrane protein 3 (VAMP3) in regulating cytokine secretions in RBL-2H3 cells, a model for MC IgE-mediated responses. In addition to VAMP3, VAMP7 is expressed in MCs, but its functional role remains elusive. The present study seeks to explore VAMP7-specific regulatory mechanisms in MCs, shedding light on one of the mechanisms governing heterogeneous secretory responses in these cells. Murine bone marrow-derived mast cells (BMMCs) were examined to analyze the subcellular distribution of inflammatory mediators, specifically TNFα, CCL2, and histamine, and VAMPs (i.e., VAMP3, VAMP7, and VAMP8). Immunocytochemistry and the transient expression of fluorescent protein-conjugated target proteins were used to discern the distribution of various inflammatory mediators and VAMP7 through confocal laser scanning microscopy. Each inflammatory mediator (TNFα, CCL2, and histamine) was found in secretory granules of different sizes within BMMCs. VAMP7 exhibited a distinct distribution compared to VAMP3 in these granules. Notably, an overlapping distribution was observed between VAMP7 and CCL2, but not between VAMP7 and TNFα or VAMP7 and histamine. This suggests that CCL2 resides within VAMP7-expressing granules and is subject to VAMP7-dependent secretory regulation. Consistently, BMMCs with VAMP7 knockdown showed markedly reduced CCL2 secretion after Ag stimulation. These observations underscore the heterogeneity of MC secretory responses and unveil a novel VAMP7-dependent CCL2 secretion mechanism within MCs. This discovery might pave the way for the development of more precise therapeutic strategies to modulate MC secretion in allergic conditions.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs.

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Mefloquine causes selective mast cell apoptosis in cutaneous mastocytosis lesions by a secretory granule-mediated pathway.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 µM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Human intelectin-2 (ITLN2) is selectively expressed by secretory Paneth cells.

Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.

Endocrine secretory granule production is caused by a lack of REST and intragranular secretory content and accelerated by PROX1.

Endocrine secretory granules (ESGs) are morphological characteristics of endocrine/neuroendocrine cells and store peptide hormones/neurotransmitters. ESGs contain prohormones and ESG-related molecules, mainly chromogranin/secretogranin family proteins. However, the precise mechanism of ESG formation has not been elucidated. In this study, we experimentally induced ESGs in the non-neuroendocrine lung cancer cell line H1299. Since repressive element 1 silencing transcription factor (REST) and prospero homeobox 1 (PROX1) are closely associated with the expression of ESG-related molecules, we edited the REST gene and/or transfected PROX1 and then performed molecular biology, immunocytochemistry, and electron and immunoelectron microscopy assays to determine whether ESG-related molecules and ESGs were induced in H1299 cells. Although chromogranin/secretogranin family proteins were induced in H1299 cells by knockout of REST and the induction was accelerated by the PROX1 transgene, the ESGs could not be defined by electron microscopy. However, a small number of ESGs were detected in the H1299 cells lacking REST and expressing pro-opiomelanocortin (POMC) by electron microscopy. Furthermore, many ESGs were produced in the REST-lacking and PROX1- and POMC-expressing H1299 cells. These findings suggest that a lack of REST and the expression of genes related to ESG content are indispensable for ESG production and that PROX1 accelerates ESG production.Trial registration: Not applicable.

Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting.

Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the TGN by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the ER. Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule sorting. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for sorting into secretory granules by self-aggregation.

Release of α-granule contents during platelet activation.

Upon activation, platelets release a plethora of factors which help to mediate their dynamic functions in hemostasis, inflammation, wound healing, tumor metastasis and angiogenesis. The majority of these bioactive molecules are released from α-granules, which are unique to platelets, and contain an incredibly diverse repertoire of cargo including; integral membrane proteins, pro-coagulant molecules, chemokines, mitogenic, growth and angiogenic factors, adhesion proteins, and microbicidal proteins. Clinically, activation of circulating platelets has increasingly been associated with various disease states. Biomarkers indicating the level of platelet activation in patients can therefore be useful tools to evaluate risk factors to predict future complications and determine treatment strategies or evaluate antiplatelet therapy. The irreversible nature of α-granule secretion makes it ideally suited as a marker of platelet activation. This review outlines the release and contents of platelet α-granules, as well as the membrane bound, and soluble α-granule cargo proteins that can be used as biomarkers of platelet activation.

Digenic Inheritance: Evidence and Gaps in Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by the inability to properly terminate an immune response. Familial HLH (FHLH) and related immune dysregulation syndromes are associated with mutations in the genes PRF1, UNC13D, STX11, STXBP2, LYST, AP3B1, and RAB27A, all of which are required for the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. Loss-of-function mutations in these genes render the cytotoxicity pathway ineffective, thereby failing to eradicate immune stimuli, such as infectious pathogens or malignant cells. The resulting persistent immune system stimulation drives hypercytokinemia, ultimately leading to severe tissue inflammation and end-organ damage. Traditionally, a diagnosis of FHLH requires the identification of biallelic loss-of-function mutations in one of these degranulation pathway genes. However, this narrow definition fails to encompass patients with other genetic mechanisms underlying degranulation pathway dysfunction. In particular, mounting clinical evidence supports a potential digenic mode of inheritance of FHLH in which single loss-of-function mutations in two different degranulation pathway genes cooperate to impair pathway activity. Here, we review the functions of the FHLH-associated genes within the degranulation pathway and summarize clinical evidence supporting a model in which cumulative defects along this mechanistic pathway may underlie HLH.

Quinacrine is not a vital fluorescent probe for vesicular ATP storage.

Quinacrine, a fluorescent amphipathic amine, has been used as a vital fluorescent probe to visualize vesicular storage of ATP in the field of purinergic signaling. However, the mechanism(s) by which quinacrine represents vesicular ATP storage remains to be clarified. The present study investigated the validity of the use of quinacrine as a vial fluorescent probe for ATP-storing organelles. Vesicular nucleotide transporter (VNUT), an essential component for vesicular storage and ATP release, is present in very low density lipoprotein (VLDL)-containing secretory vesicles in hepatocytes. VNUT gene knockout (Vnut-/-) or clodronate treatment, a VNUT inhibitor, disappeared vesicular ATP release (Tatsushima et al., Biochim Biophys Acta Molecular Basis of Disease 2021, e166013). Upon incubation of mice's primary hepatocytes, quinacrine accumulates in a granular pattern into the cytoplasm, sensitive to 0.1-µM bafilomycin A1, a vacuolar ATPase (V-ATPase) inhibitor. Neither Vnut-/- nor treatment of clodronate affected quinacrine granular accumulation. In vitro, quinacrine is accumulated into liposomes upon imposing inside acidic transmembranous pH gradient (∆pH) irrespective of the presence or absence of ATP. Neither ATP binding on VNUT nor VNUT-mediated uptake of ATP was affected by quinacrine. Consistently, VNUT-mediated uptake of quinacrine was negligible or under the detection limit. From these results, it is concluded that vesicular quinacrine accumulation is not due to a consequence of its interaction with ATP but due to ∆pH-driven concentration across the membranes as an amphipathic amine. Thus, quinacrine is not a vital fluorescent probe for vesicular ATP storage.

Identification of an antibacterial polypeptide in mouse seminal vesicle secretions.

In both men and women, pathogenic bacteria enter the reproductive tract and cause harmful symptoms. Intrauterine and oviductal inflammation after copulation may have severe effects, such as infertility, implantation failure, oviduct obstruction, and robust life-threatening bacterial infection. Human seminal plasma is considered to be protective against bacterial infection. Among its components, Semenogelin-I/-II proteins are digested to function as bactericidal factors; however, their sequences are not conserved in mammals. Therefore, alternative antibacterial (bactericidal and/or bacteriostatic) systems may exist across mammals. In this study, we examined the antibacterial activity in the seminal plasma of mice lacking a gene cluster encoding Semenogelin-I/-II counterparts. Even in the absence of the majority of seminal proteins, antibacterial activity remained in the seminal plasma. Moreover, a combination of gel chromatography and liquid chromatography coupled with tandem mass spectrometry revealed that the prostate and testis expressed 4 protein as a novel antibacterial (specifically, bacteriostatic) protein, the sequence of which is broadly conserved across mammals. Our results provide the first evidence of a bacteriostatic protein that is widely present in the mammalian seminal plasma.

In vivo Roles of Rab27 and Its Effectors in Exocytosis.

The monomeric GTPase Rab27 regulates exocytosis of a broad range of vesicles in multicellular organisms. Several effectors bind GTP-bound Rab27a and/or Rab27b on secretory vesicles to execute a series of exocytic steps, such as vesicle maturation, movement along microtubules, anchoring within the peripheral F-actin network, and tethering to the plasma membrane, via interactions with specific proteins and membrane lipids in a local milieu. Although Rab27 effectors generally promote exocytosis, they can also temporarily restrict it when they are involved in the rate-limiting step. Genetic alterations in Rab27-related molecules cause discrete diseases manifesting pigment dilution and immunodeficiency, and can also affect common diseases such as diabetes and cancer in complex ways. Although the function and mechanism of action of these effectors have been explored, it is unclear how multiple effectors act in coordination within a cell to regulate the secretory process as a whole. It seems that Rab27 and various effectors constitutively reside on individual vesicles to perform consecutive exocytic steps. The present review describes the unique properties and in vivo roles of the Rab27 system, and the functional relationship among different effectors coexpressed in single cells, with pancreatic beta cells used as an example.Key words: membrane trafficking, regulated exocytosis, insulin granules, pancreatic beta cells.

Myosin XI-B is involved in the transport of vesicles and organelles in pollen tubes of Arabidopsis thaliana.

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes, and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.