Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2023 Apr 18;28(7):471–481. doi: 10.1111/gtc.13029

Multiple pathways and independent functional pools in insulin granule exocytosis

Tetsuro Izumi 1,✉
PMCID: PMC11448364  PMID: 37070774

Abstract

In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic β cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin‐8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in β cells.

Keywords: actin cortex, diabetes, exocyst, plasma membrane, Rab27 effectors, regulated exocytosis, secretory granule, TIRF microscopy

Total internal reflection fluorescence microscopy in living pancreatic β cells lacking one or two Rab27 effectors reveals that a specific set of the effectors leads to distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis of secretory granules.

graphic file with name GTC-28-471-g001.jpg

1. GENERAL VIEWS ABOUT INSULIN GRANULE EXOCYTOSIS

In the case of synaptic vesicle exocytosis that is implemented within 1 ms after stimulation, releasable vesicles must be docked and primed on the plasma membrane in advance, because there is insufficient time for new proteins to arrive and assemble to execute these processes. Relatedly, the distance between vesicles and the Ca2+ channel that transduces Ca2+ influx upon stimulation affects their releasability because of the limited Ca2+ diffusion (Neher, 1998). By contrast, the state of the Ca2+ microdomain is less salient in secretory granule exocytosis, which occurs over much longer time courses. Even where Ca2+ concentration is abruptly upregulated by artificial caged‐Ca2+ compounds, granule exocytosis is initiated at a time point that is more than 1000‐times later than that of synaptic vesicle exocytosis (Kasai, 1999). In fact, cell capacitance analyses have revealed that insulin granule exocytosis occurs over a time course of a few seconds after depolarization stimulation. Furthermore, such initial exocytosis may not be physiologically relevant to assess the capacity of insulin secretion lasting from many minutes to hours, while nutrient absorption causes high blood glucose concentrations in vivo. Accordingly, the insulinogenic index is used clinically to measure the insulin increment in blood from 5 to 30 min after glucose load, the decrease of which is thought to represent a preferential loss of the initial phase insulin secretion in nonobese diabetes patients.

When the ambient glucose concentration is abruptly increased, isolated pancreatic islets display biphasic insulin secretion: a rapid peak within a few minutes followed by a sustained lower level thereafter (the first and the second phases, respectively). However, there is no strong consensus regarding the mechanism of this biphasic time course. Some have suggested that the mechanism involves the biphasic rise in Ca2+ concentration with possible involvement of additional amplifying signals in the second phase (Henquin et al., 2002), although this has not been well characterized. Others have postulated two compartments of granules, based on findings from total internal reflection fluorescence (TIRF) microscopy in living β cells expressing cargo protein fused with fluorescent protein. They suggest that the first phase reflects activity of granules predocked or closely located to the plasma membrane, whereas the second phase reflects that of granules newly recruited from the cell interior (Ohara‐Imaizumi & Nagamatsu, 2006; Seino et al., 2011). However, it is important to note that both phases are separated over a time course of several minutes, which affords time for granules to be translocated from the cell interior even in the first few minutes after glucose stimulation. Furthermore, given that biphasic insulin secretion occurs only after preincubation in a low‐glucose condition, the simplest explanation may be that the peak in the first phase simply reflects the accumulation of granules in a fusion‐ready state during the preceding unstimulated period, whereas the sustained level in the second phase represents the exocytic rate in equilibrium at which granules become fusion‐ready during sustained stimulation. In this model, no differential signals or distinct granule pools need to be postulated to explain the biphasic secretion. Below, it will be shown that such a long time course permits granule exocytosis in heterogeneous manners and via different routes.

TIRF microscopy enables visualization and monitoring of secretory granules locating within an evanescent field, which is 100–200 nm from the plasma membrane, and reveals heterogeneous prefusion behaviors of insulin granules. Fusion occurs from granules visible before stimulation, which we call “residents,” those becoming visible during stimulation, which we call “visitors,” and those only visible at the instant of fusion, which we call “passengers.” (Kasai et al., 2008; see TIRF microscopic images in Figure 1a) Because all of these types show similar time courses and amplitudes of fluorescent intensity changes during fusion reactions on average, most of them likely represent single granule exocytosis. Although the resident type exocytosis tends to be predominant in the initial phase of exocytosis, a common finding from different laboratories is that heterogeneous exocytosis can occur at any time point after stimulation (Kasai et al., 2008; Ohara‐Imaizumi et al., 2004; Shibasaki et al., 2007; Xie et al., 2012). For example, the exocytosis from granules residing beneath the plasma membrane continues during glucose stimulation, whereas that from newly recruited granules occurs within the first few minutes (see examples in Figure 1b). Furthermore, although the resident type exocytosis is often thought to be derived from granules docked to the plasma membrane, it can actually be divided into two subclasses that from immobile granules molecularly tethered to the plasma membrane and that from diffusible granules just locating nearby, which display differential fusion probabilities (Mizuno et al., 2016). Thus, it is not the case that exocytosis occurs from predocked granules exclusively, preferentially, or even antecedently.

FIGURE 1.

FIGURE 1

Open in a new tab

Examples of insulin granule exocytosis in a single beta cell by total internal reflection fluorescence (TIRF) microscopy. A monolayer of islet cells from C57BL/6N mice (n = 12 cells from three mice) were infected with adenovirus encoding insulin‐enhanced green fluorescent protein (EGFP) and were observed by TIRF microscopy. The penetration depth of the evanescent field was 100 nm. (a) Images were acquired at 103‐ms intervals. Insulin granule fusion events with a flash followed by diffusion of EGFP signals in response to 25 mmol/L glucose for 20 min were manually selected and assigned to one of three types: residents, which were visible for more than 10 s prior to fusion; visitors, which became visible within 10 s of fusion; and passengers, which were not visible prior to fusion. The red boxes indicate the time of the beginning of fusion. (b) The histograms show the numbers of fusion events per 200 μm2 at 1‐min intervals in each single cell (red, residents; light green, visitors; dark green, passengers).

Although simple TIRF microscopy monitoring of fluorescent granule cargo protein suggests that the granule exocytic pathway is neither single nor linear, but rather multilateral, it is incapable of revealing the underlying mechanism. However, based on their morphological behavior prior to fusion, granules appear to be stalled in different exocytotic states before stimulation in each type of exocytosis. This suggests the existence of differential rate‐limiting and/or fusion‐inhibitory steps in regulated exocytosis. In that case, the molecular mechanism for stimulus‐induced liberation from these steps must vary among different types of exocytosis. The following sections describe the principal molecules involved in these regulatory steps, which include monomeric GTPase, Rab27a, and its multiple effectors locating on the granule membrane (Izumi, 2021).

2. MOLECULAR MECHANISMS OF INSULIN GRANULE ACCUMULATION AT THE CELL PERIPHERY

For secretory granules to fuse with the plasma membrane, they must first be localized at the cell periphery. In fact. they are not evenly distributed in the cytoplasm, but are enriched in the actin cortex, a peripheral microfilament web beneath the plasma membrane that maintains the cell's shape and integrity, and along the plasma membrane.

2.1. Granule anchoring in the actin cortex

In the pheochromocytoma cell line, PC12, fluorescence‐labeled, nascent granules generated at the trans‐Golgi network are transported in a microtubule‐dependent manner within a few seconds to the cell periphery and are captured in the actin cortex where maturation then proceeds over several hours (Rudolf et al., 2001). The anchoring of secretory granules within the actin cortex is executed by one of the Rab27 effectors, exophilin‐8 (also known as MyRIP or Slac2c; approved human gene symbol, MYRIP) (Bierings et al., 2012; Desnos et al., 2003; Huet et al., 2012; Mizuno et al., 2011; Nightingale et al., 2009). Exophilin‐8 does so by connecting Rab27a on granules with RIM‐BP2 and the actin motor protein, myosin‐VIIa (Fan et al., 2017). In fact, knockdown of exophilin‐8, RIM‐BP2, or myosin‐VIIa redistributes insulin granules almost evenly in the cytoplasm and decreases their exocytosis in the β‐cell line, INS‐1. However, the absence of exophilin‐8 does not decrease the number of granules visible by the TIRF microscopy (Zhao et al., 2023), which challenges the existing model that granules anchored within the actin cortex supply granules residing beneath the plasma membrane. Furthermore, although exophilin‐8 deficiency markedly decreases both the resident and the passenger types of exocytosis, it does not block insulin secretion. These findings indicate that a significant number of granules can reach and fuse with the plasma membrane without prior capture in the actin cortex. Indeed, in chromaffin cells, secretagogue‐induced, Ca2+‐dependent disassembly of cortical actin structures form open spaces devoid of F‐actin in the cytoplasm and channel‐like structures perpendicular to plasma membrane conducting granules to the cell limits (Giner et al., 2005). Furthermore, in contrast to the case in β cells, the depletion of exophilin‐8 in human umbilical vein endothelial cells (HUVECs) facilitates granule passage through the actin network and increases both basal and stimulated secretion of von Willebrand factor (VWF), despite the loss of peripheral Weibel–Palade bodies (Nightingale et al., 2009). Thus, granule anchoring within the actin cortex has multiple effects on net secretion among different secretory cells. It appears to play dual roles for granule exocytosis in clustering granules in the cell periphery and in blocking their access to the plasma membrane.

2.2. Granule docking to the plasma membrane

Under electron microscopy, a substantial portion of insulin granules (10%–15%) are directly attached to the plasma membrane. Such docked granules are almost completely lost in β cells lacking another Rab27 effector, granuphilin (also known as exophilin‐2 or Slp4; approved human gene symbol, SYTL4) (Gomi et al., 2005). Despite the docking defect, granuphilin deficiency enhances both basal and stimulated insulin secretion (Gomi et al., 2005; Kasai et al., 2008). These findings may be counterintuitive if docking is thought as a necessary prior step for fusion. However, in contrast to constitutive exocytosis where vesicles attached to the plasma membrane automatically fuse without stimulation, regulated exocytosis must include a temporal constraint that prevents spontaneous fusing of the vesicles beneath the plasma membrane (Izumi, 2011). In fact, granuphilin both mediates stable docking and simultaneously limits fusion of insulin granules by interacting with the closed form of syntaxin‐1–3 (Torii et al., 2002; Torii et al., 2004; Wang et al., 2011), which is incapable of forming fusion machinery with other SNARE proteins (Misura et al., 2000). Therefore, it is conceivable that granule docking does not involve the assembly of SNARE proteins from vesicular and plasma membranes. Consistent with this view, two‐photon fluorescence lifetime imaging shows that, although vesicles in presynaptic terminals preassemble SNARE proteins, insulin granules do not preassemble them before stimulation (Takahashi et al., 2015). By TIRF microscopy, granuphilin deficiency markedly increases all three types of exocytosis, although the number of visible granules is markedly decreased (Zhao et al., 2023). It seems that the absence of granules molecularly tethered to the plasma membrane facilitates access of incoming granules to the fusion machinery. Furthermore, the absence of granuphilin may also increase the fusion‐competent, open form of syntaxins on the plasma membrane.

TIRF microscopy reveals that 80%–85% of the visible granules are granuphilin‐positive and immobile in a resting state in the β‐cell line, MIN6, likely reflecting that they are physically tethered to the plasma membrane (Mizuno et al., 2016). Nevertheless, granuphilin‐positive granules and the remaining granuphilin‐negative, mobile granules fuse with comparable frequencies in response to depolarization stimulation, which means that the fusion probability of the positive granules is less than 20% of that of the negative granules (note that “granuphilin‐negative” granules by TIRF microscopy simply denote an absence of granuphilin within an evanescent field, such as in the hemisphere closest to the plasma membrane, even though granuphilin may be present in the opposite hemisphere). Thus, the molecularly tethered (docked) granules may be fusion‐reluctant compared with untethered granules below the plasma membrane. However, this does not mean that granuphilin causes “dead‐end docking,” as suggested previously (Verhage & Sorensen, 2008), but instead mediates fusion‐capable, functional docking. For these docked granules to fuse, granuphilin‐associated syntaxins must be converted from the closed form to the open form to assemble the SNARE proteins. In synaptic vesicle exocytosis, Munc13 family proteins convert the closed form of syntaxins into the open form for fusion (Richmond et al., 2001). In line with this, Munc13‐2 has recently been suggested to accumulate stimulus‐dependently on granuphilin‐positive granules just prior to their fusion (Mizuno & Izumi, 2022). As found for granule anchoring within the actin cortex by exophilin‐8, granule docking to the plasma membrane by granuphilin has dual roles in accumulating granules beneath the plasma membrane and in imposing a fusion‐constraint on those docked granules. Again, in contrast to the case in β cells, depletion of granuphilin in HUVECs decreases stimulus‐evoked VWF secretion (Bierings et al., 2012). It seems that the major gating step to limit fusion in regulated exocytosis differs among cells: there is granuphilin‐mediated granule docking to the plasma membrane in β cells, while there is exophilin‐8‐mediated granule anchoring within the actin cortex in HUVECs.

3. MOLECULAR MECHANISM OF MULTIPLE INSULIN GRANULE EXOCYTIC PATHWAYS

3.1. Exocytosis of granules residing beneath the plasma membrane prior to stimulation

To examine the functional relationship between granule anchoring within the actin cortex and docking to the plasma membrane in exocytosis, we generated exophilin‐8/granuphilin double knockout mice. The simultaneous absence of the two effectors almost completely suppressed the increased levels of the passenger and the visitor types of exocytosis found in granuphilin singly deficient cells to the decreased levels found in exophilin‐8 singly deficient cells (Zhao et al., 2023). This suggests that these types of exocytosis are largely derived from granules within the actin cortex (see details in Figure 2b). In contrast, the double deficiency did not affect the increase in the resident type exocytosis found in granuphilin singly deficient cells. These findings indicate that granules can arrive close to the plasma membrane and fuse efficiently without prior capture in the actin cortex by exophilin‐8 or stable docking to the plasma membrane by granuphilin, which challenges the prevailing concept that both steps are involved in forming a reserve pool and a ready releasable pool, respectively.

FIGURE 2.

FIGURE 2

Open in a new tab

Molecular mechanism of multiple insulin granule exocytic pathways. (a) Exocytosis of granules residing beneath the plasma membrane prior to stimulation. Granules are stably docked to the plasma membrane by granuphilin. They can fuse after the granuphilin‐associated, closed form of syntaxin 1 is converted into the open form, which is thought to be mediated by exophilin‐8‐associated priming factors such as RIM‐BP2. (b) Exocytosis of granules mobilized to the plasma membrane after stimulation. Granules are anchored in the actin cortex by exophilin‐8. They fuse either immediately (right: the passenger type) or after remaining beneath the plasma membrane (left: the visitor type), once they are released from the actin network by stimulation. The former granules fuse immediately via the direct interaction of melanophilin on the granule membrane with the fusion‐competent, open form of syntaxin‐4 on the plasma membrane.

It is notable that, although exophilin‐8 deficiency does not decrease the resident type exocytosis in the absence of granuphilin, it does so in the presence of granuphilin. To directly assess the effect of exophilin‐8 deficiency on the exocytosis of granuphilin‐mediated, docked granules, we expressed fluorescently labeled granuphilin in granuphilin/exophilin‐8 doubly deficient cells and in granuphilin singly deficient cells to mimic exophilin‐8 singly deficient cells and wild‐type cells, respectively. We found that the level of the resident type exocytosis from granuphilin‐positive granules was almost completely inhibited by the absence of exophilin‐8 (Zhao et al., 2023). As described elsewhere, granuphilin‐mediated, docked granules are thought to require priming factors for fusion that convert the associated, closed form of syntaxin into the open form. Exophilin‐8‐associated RIM‐BP2 and RIM‐BP2‐associated RIM and Munc13 are known to have such a priming role in synaptic vesicle exocytosis (Brockmann et al., 2019; Brockmann et al., 2020). In fact, the exophilin‐8 mutant that cannot interact with RIM‐BP2 fails to rescue the decreased insulin secretion in exophilin‐8 deficient cells (Fan et al., 2017). These exophilin‐8‐associated priming factors could also act on syntaxin not associated with granuphilin, because exophilin‐8 deficiency also decreases the resident type exocytosis from granuphilin‐negative granules to some extent.

3.2. Exocytosis of granules mobilized to the plasma membrane after stimulation

The passenger type exocytosis observed by TIRF microscopy, in which granules immediately fuse once entering the evanescent field, suggests the possibility of exocytosis bypassing a stable docking step. However, the current understanding of ultrafast synaptic vesicle exocytosis does not include the specific mechanism for undocked vesicle exocytosis. Of course, the absence of either a molecular or simply hypothetical mechanism has led some investigators to doubt the existence of any such mechanism. For example, the absence of any such mechanism was reported in human β cells expressing neuropeptide Y fused with mCherry (Gandasi et al., 2018). However, because the passenger type is visible only in one frame (103 ms) as a flash by granule neutralization during the fusion, it was later found to be difficult to find when granules are visualized by pH‐insensitive mCherry (Wang et al., 2020). Instead, when granules were labeled using the pH‐sensitive enhanced green fluorescent protein (EGFP), this type of exocytosis was detected in both mouse and human β cells.

It has recently been shown that the Rab27 effector, melanophilin (also known as exophilin‐3 or Slac2a; approved human gene symbol, MLPH), is specifically involved in the passenger type exocytosis. Melanophilin is known to link melanosomes to actin filaments in the cell periphery via its affinity to myosin‐Va in skin melanocytes, which enables melanosomes to be transferred to neighboring keratinocytes (Hammer 3rd & Sellers, 2011). Although both melanophilin and myosin‐Va are expressed in β cells, silencing them cannot affect the peripheral localization of insulin granules by light microscopy (Fan et al., 2017; Wang et al., 2020). Therefore, in contrast to the exophilin‐8/myosin‐VIIa complex, the melanophilin/myosin‐Va complex is not involved in granule clustering within the actin cortex. However, analyses in melanophilin‐deficient β cells have revealed a specific decrease in the passenger type exocytosis (Wang et al., 2020). In contrast to granuphilin, melanophilin interacts with the open form of syntaxin‐4 that is able to assemble with other SNARE proteins. Furthermore, with a stimulus‐induced, intracellular Ca2+ increase, melanophilin dissociates from myosin‐Va and actin and instead associates with syntaxin‐4. These findings are consistent with the model, in which melanophilin‐associated granules are released from the actin cortex and fuse only after stimulation in the passenger type exocytosis (Figure 2b). However, the mechanism by which those granules released into the cytoplasm are directed to syntaxin‐4 on the plasma membrane remains unknown. The actin tracks are unlikely to be involved in this mobilization, because they are generally organized parallel to, rather than perpendicular to, the plasma membrane in animal cells (Hammer 3rd & Sellers, 2011). Furthermore, based on the morphological definition of the passenger type exocytosis, those granules must pass the evanescent field measuring 100–200 nm, and must do so within 100 ms, which would require a velocity of 1–2 μm/s. That would be faster than would be possible with myosin‐Va, one of the fastest processive myosins, which can run at the maximum velocity of 350–550 nm/s even in a cell‐free system (Baker et al., 2004; Cheney et al., 1993). Alternatively, granules released from the actin network upon stimulation may gain access to microtubule tracks to move by faster kinesin motors at a velocity of ~1 μm/s (Coy et al., 1999). However, it is more likely that a multi‐subunit protein complex, the exocyst, which physically links exophilin‐8 and melanophilin, is involved in this process, as described below.

3.3. The exocyst connects different Rab27 effectors

TIRF microscopy cannot precisely localize granules showing the passenger type exocytosis prior to stimulation. However, they are thought to be derived from the actin cortex, because depletion of exophilin‐8 or melanophilin, both of which interact with the actin motor proteins, myosins, markedly reduces this type of exocytosis. To examine the functional relationship between the two effectors, we generated mice doubly deficient in exophilin‐8 and melanophilin. These β cells show a phenocopy of exophilin‐8 singly deficient cells: while the number of granules beneath the plasma membrane is not changed, both the resident and the passenger types of exocytosis are decreased to the levels found in exophilin‐8 singly deficient cells (Zhao et al., 2023). Considering that melanophilin singly deficient cells only show a decrease in the passenger type, exophilin‐8 appears to function upstream of melanophilin.

Because single granules can harbor multiple Rab27a molecules, different effectors could function in sequence on the same granules, as suggested previously (Izumi, 2021). However, we do not understand the transition mechanism by which granules accumulated within a relatively broad area of the actin cortex by exophilin‐8 are mobilized to the cell limits to enable melanophilin‐mediated fusion. The activity‐driven relaxation of the cortical actomyosin II may contribute to this process, because granules embedded in the cortical actin network undergo a synchronized transition towards the plasma membrane in response to secretagogues in chromaffin cells (Papadopulos et al., 2015). However, it remains unknown just how melanophilin on granules released into the cytoplasm efficiently interacts with syntaxin‐4 on the plasma membrane. We found that exophilin‐8 and melanophilin interact through the exocyst (Zhao et al., 2023), which is known to physically tether secretory vesicles to the plasma membrane in a broad range of secretory pathways, including constitutive exocytosis (Wu & Guo, 2015). Knockdown of the exocyst subunit in wild‐type cells decreases both the resident and passenger types of exocytosis, as found in exophilin‐8‐deficient cells. In contrast, the exocyst knockdown induces no additional decrease in either type of exocytosis in exophilin‐8‐deficient cells. These findings suggest that the exocyst functions downstream of exophilin‐8. Considering its universal function, the exocyst likely tethers granules to the plasma membrane to facilitate the complex formation between melanophilin and syntaxin‐4 in the passenger type exocytosis.

3.4. Coordination among different Rab27 effectors and the exocyst

Based on observations of the exocytic phenotypes by TIRF microscopy of β cells deficient in Rab27 effectors and/or the exocyst, we propose that there are at least four distinguishable types of insulin granule exocytosis (Figure 3). Granules captured in the actin cortex by exophilin‐8 fuse immediately once released into the cytoplasm after stimulation via the interaction of melanophilin on the granule membrane with the open form of syntaxin‐4 on the plasma membrane (the passenger type). A relatively small portion of granules released from the actin cortex remain beneath the plasma membrane for a period prior to fusion (the visitor type). The finding that knockdown of the exocyst subunit eliminates the increase in these types of exocytosis in granuphilin‐deficient cells suggests that the exocyst helps tether these granules to the plasma membrane for fusion (Zhao et al., 2023). Other granules escaping the anchoring in the actin cortex are either stably docked to the plasma membrane by granuphilin (the resident type A) or remain untethered beneath the plasma membrane prior to fusion (the resident type B). The former immobilized granules fuse after granuphilin‐associated syntaxins are converted into the fusion‐competent, open form, possibly by exophilin‐8‐associated priming factors such as RIM‐BP2. The latter mobile granules fuse stochastically after being directed to the plasma membrane by the exocyst. The exocyst may also facilitate the complex formation between granuphilin and syntaxins‐1–3 during the granule docking process. However, it should be noted that silencing of the exocyst has no influence on the exocytosis of granules that have already been immobilized to the plasma membrane by granuphilin (Zhao et al., 2023). There is no evidence suggesting that granules showing the resident type exocytosis are derived from granules previously anchored in the actin cortex, because the number of granules residing beneath the plasma membrane is not affected by exophilin‐8 deficiency. It should be noted, however, that granule exocytosis is still present in β cells lacking any of the single or double Rab27 effectors, which suggests the existence of a default, constitutive‐like exocytic pathway. In general, vesicles can be delivered to the cell periphery via actin tracks by myosin motors and Rab‐interacting proteins, albeit slowly (Alzahofi et al., 2020; Schuh, 2011). Furthermore, SNAREs in native plasma membranes are constitutively active even if not engaged in fusion events in PC12 cells (Lang et al., 2002). Given these parallel secretory pathways, functional granule pools, such as a reserve pool (RP) and a readily releasable pool (RRP), cannot be defined singly in either morphological or molecular terms (Figure 3). These different pools mediate fusion in parallel after stimulation and contribute to both the first and the second phases of glucose‐stimulated insulin secretion.

FIGURE 3.

FIGURE 3

Open in a new tab

Functional granule pools for different types of insulin granule exocytosis. At least four types of insulin granule exocytosis function in parallel, the resident type A, the resident type B, the visitor type, and the passenger type (see text). The exocyst also facilitates the tethering of granules to the plasma membrane. Given these parallel secretory pathways, functional granule pools, such as a reserve pool (RP) and a readily releasable pool (RRP), cannot be defined singly in either morphological or molecular terms. For example, granules captured in the actin cortex are thought to be fusion‐ready in the passenger type exocytosis (RRP1) but not yet so in the visitor type exocytosis (RP1), respectively. Furthermore, granules stably docked to the plasma membrane by granuphilin may be fusion‐ready once they are primed (RRP2) but may be fusion‐reluctant if the associated syntaxin remains in the closed form (RP2). It is currently unknown whether the priming reaction occurs before or after stimulation. Furthermore, it should be realized that granules locating beneath the plasma membrane without being tethered by granuphilin can also fuse after stimulation with the aid of the exocyst and, thus, comprise a readily releasable pool (RRP3). However, the granule cluster sites, such as in the actin cortex and along the plasma membrane, may simply reflect the presence of critical regulatory steps that prevent spontaneous or excessive fusion.

It is now clear that multiple Rab27 effectors regulate distinct exocytic steps in the same cell. However, the specific mechanism by which these steps are regulated in a coordinated manner for efficient exocytosis is unknown. It is known that the majority of Rab27 exists in a GTP‐bound form in many cells, including β cells (Yi et al., 2002). Fluorescence recovery after photobleaching analyses indicates that Rab27 and its effectors show low turnover and stable localization on the vesicle membrane (Handley et al., 2007; Handley & Burgoyne, 2008). It seems that global GTP hydrolysis of Rab27 occurs only after the final granule fusion with the plasma membrane in stimulated platelets (Kondo et al., 2006). Thus, it is unlikely that the effector compositions on each granule are shifted in a stepwise manner during membrane trafficking. In fact, all Rab27 effectors previously examined in β cells, including Noc2 that interacts with Rab2a located in the perinuclear region as well as Rab27 on granules, are distributed on insulin granules throughout the cytoplasm (Fan et al., 2017; Matsunaga et al., 2017; Wang et al., 2020; Yi et al., 2002). Distinct Rab27 effectors on the same granule appear to advance exocytic processes through sequential interactions with specific proteins and lipids existing in a surrounding local milieu (Izumi, 2021).

4. POSSIBLE DEFECTS IN INSULIN GRANULE EXOCYTOSIS IN DIABETES

Although ashen mice that harbor a loss‐of‐function mutation of the Rab27a gene display a marked decrease in the glucose‐stimulated insulin secretion (Kasai et al., 2005), it is currently unknown whether human patients of Griscelli syndrome type 2 with mutation of the Rab27a gene have impaired insulin secretion and/or glucose intolerance. These patients die young due to immunodeficiency, unless bone marrow transplantation is successful (Van Gele et al., 2009). In the presence of such serious symptoms, it may be difficult to scrutinize the abnormal secretion in β cells. No mutations of Rab27 effectors have been reported either in humans with impaired insulin secretion and/or glucose intolerance. However, given that multiple exocytic pathways function in parallel in β cells and that Rab27 effectors are thought to play regulatory roles rather than essential roles in granule exocytosis, it is likely that the mutation displays only partial secretory defects, as found in murine β cells deficient in them.

It was reported that the number of morphologically docked granules and their exocytic events are reduced in isolated β cells of human type 2 patients, based on TIRF microscopic analyses (Gandasi et al., 2018). However, it is unknown whether the defect is specific to docked granule exocytosis, because that study failed to monitor the passenger type of undocked granule exocytosis using pH‐insensitive NPY‐mCherry, as described previously. It was also reported that the number of granules visible by TIRF microscopy is decreased in β cells of the Goto‐Kakizaki diabetic rat, although the number of the total or remaining “undocked” granules is not affected (Ohara‐Imaizumi et al., 2004). However, it should be realized that most undocked granules in the deep cell interior cannot fuse, simply due to their physical distance from the plasma membrane. Furthermore, because, at the present time, we cannot morphologically characterize the precise original location of granules that undergo the passenger or visitor type of exocytosis, such releasable undocked granules at the cell periphery may have also been decreased. Given that diabetes may arise with marked genetic heterogeneity, the decrease in docked granule exocytosis may not be inherent to the pathogenesis of diabetes, but may instead reflect increased exocytosis under the condition of prolonged hyperglycemia in this disease. The specificity of the decrease in docked granule exocytosis in diabetic states needs to be carefully evaluated.

5. CONCLUSIONS

In summary, the TIRF microscopic findings in cells deficient in Rab27 effectors have provided useful information about their functional hierarchy and the distinct rate‐limiting steps in multiple granule exocytic pathways. Future research will investigate the transitional mechanism between the steps by which the different effectors function. Although it has traditionally been considered that granules docked to the plasma membrane form a readily releasable pool and that those accumulated within the actin cortex form a reserve pool, it is crucial to recognize the absence of direct evidence suggesting that granules anchored within the actin cortex supply those docked to the plasma membrane. Such granule clustering sites appear to represent the presence of gating steps to prevent unregulated fusion. Therefore, granules showing different types of exocytosis most likely await stimulation at a distinct exocytic stage and should form an independent reserve and readily releasable pool, if any (Figure 3).

FUNDING INFORMATION

The author received research support from Eli Lilly Japan K.K. and Teijin Pharma Donation.

CONFLICT OF INTEREST STATEMENT

The author declares no conflict of interest.

ACKNOWLEDGMENTS

I would like to thank Kunli Zhao and Kouichi Mizuno for preparing Figure 1. I also thank Sachiko Shigoka for help to prepare Figure 3 and the manuscript. I appreciate the contribution of scientific findings from the previous and present members in my laboratory.

Izumi, T. (2023). Multiple pathways and independent functional pools in insulin granule exocytosis. Genes to Cells, 28(7), 471–481. 10.1111/gtc.13029

Communicated by: Eisuke Nishida

REFERENCES

  1. Alzahofi, N. , Welz, T. , Robinson, C. L. , Page, E. L. , Briggs, D. A. , Stainthorp, A. K. , Reekes, J. , Elbe, D. A. , Straub, F. , Kallemeijn, W. W. , Tate, E. W. , Goff, P. S. , Sviderskaya, E. V. , Cantero, M. , Montoliu, L. , Nedelec, F. , Miles, A. K. , Bailly, M. , Kerkhoff, E. , & Hume, A. N. (2020). Rab27a co‐ordinates actin‐dependent transport by controlling organelle‐associated motors and track assembly proteins. Nature Communications, 11(1), 3495. 10.1038/s41467-020-17212-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker, J. E. , Krementsova, E. B. , Kennedy, G. G. , Armstrong, A. , Trybus, K. M. , & Warshaw, D. M. (2004). Myosin V processivity: Multiple kinetic pathways for head‐to‐head coordination. Proceedings of the National Academy of Sciences of the United States of America, 101(15), 5542–5546. 10.1073/pnas.0307247101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bierings, R. , Hellen, N. , Kiskin, N. , Knipe, L. , Fonseca, A. V. , Patel, B. , Meli, A. , Rose, M. , Hannah, M. J. , & Carter, T. (2012). The interplay between the Rab27A effectors Slp4‐a and MyRIP controls hormone‐evoked Weibel–Palade body exocytosis. Blood, 120(13), 2757–2767. 10.1182/blood-2012-05-429936 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brockmann, M. M. , Maglione, M. , Willmes, C. G. , Stumpf, A. , Bouazza, B. A. , Velasquez, L. M. , Grauel, M. K. , Beed, P. , Lehmann, M. , Gimber, N. , Schmoranzer, J. , Sigrist, S. J. , Rosenmund, C. , & Schmitz, D. (2019). RIM‐BP2 primes synaptic vesicles via recruitment of Munc13‐1 at hippocampal mossy fiber synapses. eLife, 8, e43243. 10.7554/eLife.43243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brockmann, M. M. , Zarebidaki, F. , Camacho, M. , Grauel, M. K. , Trimbuch, T. , Sudhof, T. C. , & Rosenmund, C. (2020). A trio of active zone proteins comprised of RIM‐BPs, RIMs, and Munc13s governs neurotransmitter release. Cell Reports, 32(5), 107960. 10.1016/j.celrep.2020.107960 [DOI] [PubMed] [Google Scholar]
  6. Cheney, R. E. , O'Shea, M. K. , Heuser, J. E. , Coelho, M. V. , Wolenski, J. S. , Espreafico, E. M. , Forscher, P. , Larson, R. E. , & Mooseker, M. S. (1993). Brain myosin‐V is a two‐headed unconventional myosin with motor activity. Cell, 75(1), 13–23. [DOI] [PubMed] [Google Scholar]
  7. Coy, D. L. , Wagenbach, M. , & Howard, J. (1999). Kinesin takes one 8‐nm step for each ATP that it hydrolyzes. The Journal of Biological Chemistry, 274(6), 3667–3671. 10.1074/jbc.274.6.3667 [DOI] [PubMed] [Google Scholar]
  8. Desnos, C. , Schonn, J. S. , Huet, S. , Tran, V. S. , el ‐Amraoui, A. , Raposo, G. , Fanget, I. , Chapuis, C. , Ménasché, G. , de Saint Basile, G. , Petit, C. , Cribier, S. , Henry, J. P. , & Darchen, F. (2003). Rab27A and its effector MyRIP link secretory granules to F‐actin and control their motion towards release sites. The Journal of Cell Biology, 163(3), 559–570. 10.1083/jcb.200302157 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fan, F. , Matsunaga, K. , Wang, H. , Ishizaki, R. , Kobayashi, E. , Kiyonari, H. , Mukumoto, Y. , Okunishi, K. , & Izumi, T. (2017). Exophilin‐8 assembles secretory granules for exocytosis in the actin cortex via interaction with RIM‐BP2 and myosin‐VIIa. eLife, 6, e26174. 10.7554/eLife.26174 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gandasi, N. R. , Yin, P. , Omar‐Hmeadi, M. , Ottosson Laakso, E. , Vikman, P. , & Barg, S. (2018). Glucose‐dependent granule docking limits insulin secretion and is decreased in human type 2 diabetes. Cell Metabolism, 27(2), 470.e4–478.e4. 10.1016/j.cmet.2017.12.017 [DOI] [PubMed] [Google Scholar]
  11. Giner, D. , Neco, P. , Frances Mdel, M. , Lopez, I. , Viniegra, S. , & Gutierrez, L. M. (2005). Real‐time dynamics of the F‐actin cytoskeleton during secretion from chromaffin cells. Journal of Cell Science, 118(Pt 13), 2871–2880. 10.1242/jcs.02419 [DOI] [PubMed] [Google Scholar]
  12. Gomi, H. , Mizutani, S. , Kasai, K. , Itohara, S. , & Izumi, T. (2005). Granuphilin molecularly docks insulin granules to the fusion machinery. The Journal of Cell Biology, 171(1), 99–109. 10.1083/jcb.200505179 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hammer, J. A., 3rd , & Sellers, J. R. (2011). Walking to work: Roles for class V myosins as cargo transporters. Nature Reviews. Molecular Cell Biology, 13(1), 13–26. 10.1038/nrm3248 [DOI] [PubMed] [Google Scholar]
  14. Handley, M. T. , & Burgoyne, R. D. (2008). The Rab27 effector rabphilin, unlike granuphilin and Noc2, rapidly exchanges between secretory granules and cytosol in PC12 cells. Biochemical and Biophysical Research Communications, 373(2), 275–281. 10.1016/j.bbrc.2008.06.043 [DOI] [PubMed] [Google Scholar]
  15. Handley, M. T. , Haynes, L. P. , & Burgoyne, R. D. (2007). Differential dynamics of Rab3A and Rab27A on secretory granules. Journal of Cell Science, 120(Pt 6), 973–984. 10.1242/jcs.03406 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henquin, J. C. , Ishiyama, N. , Nenquin, M. , Ravier, M. A. , & Jonas, J. C. (2002). Signals and pools underlying biphasic insulin secretion. Diabetes, 51(Suppl 1), S60–S67. 10.2337/diabetes.51.2007.s60 [DOI] [PubMed] [Google Scholar]
  17. Huet, S. , Fanget, I. , Jouannot, O. , Meireles, P. , Zeiske, T. , Larochette, N. , Darchen, F. , & Desnos, C. (2012). Myrip couples the capture of secretory granules by the actin‐rich cell cortex and their attachment to the plasma membrane. The Journal of Neuroscience, 32(7), 2564–2577. 10.1523/JNEUROSCI.2724-11.2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Izumi, T. (2011). Heterogeneous modes of insulin granule exocytosis: Molecular determinants. Frontiers in Bioscience, 16, 360–367. 10.2741/3692 [DOI] [PubMed] [Google Scholar]
  19. Izumi, T. (2021). In vivo roles of Rab27 and its effectors in exocytosis. Cell Structure and Function, 46(2), 79–94. 10.1247/csf.21043 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kasai, H. (1999). Comparative biology of Ca2+‐dependent exocytosis: Implications of kinetic diversity for secretory function. Trends in Neurosciences, 22(2), 88–93. 10.1016/s0166-2236(98)01293-4 [DOI] [PubMed] [Google Scholar]
  21. Kasai, K. , Fujita, T. , Gomi, H. , & Izumi, T. (2008). Docking is not a prerequisite but a temporal constraint for fusion of secretory granules. Traffic, 9(7), 1191–1203. 10.1111/j.1600-0854.2008.00744.x [DOI] [PubMed] [Google Scholar]
  22. Kasai, K. , Ohara‐Imaizumi, M. , Takahashi, N. , Mizutani, S. , Zhao, S. , Kikuta, T. , Kasai, H. , Nagamatsu, S. , Gomi, H. , & Izumi, T. (2005). Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation. The Journal of Clinical Investigation, 115(2), 388–396. 10.1172/JCI22955 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kondo, H. , Shirakawa, R. , Higashi, T. , Kawato, M. , Fukuda, M. , Kita, T. , & Horiuchi, H. (2006). Constitutive GDP/GTP exchange and secretion‐dependent GTP hydrolysis activity for Rab27 in platelets. The Journal of Biological Chemistry, 281(39), 28657–28665. 10.1074/jbc.M603227200 [DOI] [PubMed] [Google Scholar]
  24. Lang, T. , Margittai, M. , Holzler, H. , & Jahn, R. (2002). SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs. The Journal of Cell Biology, 158(4), 751–760. 10.1083/jcb.200203088 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matsunaga, K. , Taoka, M. , Isobe, T. , & Izumi, T. (2017). Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2. Journal of Cell Science, 130(3), 541–550. 10.1242/jcs.195479 [DOI] [PubMed] [Google Scholar]
  26. Misura, K. M. , Scheller, R. H. , & Weis, W. I. (2000). Three‐dimensional structure of the neuronal‐Sec1‐syntaxin 1a complex. Nature, 404(6776), 355–362. 10.1038/35006120 [DOI] [PubMed] [Google Scholar]
  27. Mizuno, K. , Fujita, T. , Gomi, H. , & Izumi, T. (2016). Granuphilin exclusively mediates functional granule docking to the plasma membrane. Scientific Reports, 6, 23909. 10.1038/srep23909 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mizuno, K. , & Izumi, T. (2022). Munc13b stimulus‐dependently accumulates on granuphilin‐mediated, docked granules prior to fusion. Cell Structure and Function, 47(1), 31–41. 10.1247/csf.22005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mizuno, K. , Ramalho, J. S. , & Izumi, T. (2011). Exophilin8 transiently clusters insulin granules at the actin‐rich cell cortex prior to exocytosis. Molecular Biology of the Cell, 22(10), 1716–1726. 10.1091/mbc.E10-05-0404 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Neher, E. (1998). Vesicle pools and Ca2+ microdomains: New tools for understanding their roles in neurotransmitter release. Neuron, 20(3), 389–399. 10.1016/s0896-6273(00)80983-6 [DOI] [PubMed] [Google Scholar]
  31. Nightingale, T. D. , Pattni, K. , Hume, A. N. , Seabra, M. C. , & Cutler, D. F. (2009). Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells. Blood, 113(20), 5010–5018. 10.1182/blood-2008-09-181206 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ohara‐Imaizumi, M. , & Nagamatsu, S. (2006). Insulin exocytotic mechanism by imaging technique. Journal of Biochemistry, 140(1), 1–5. 10.1093/jb/mvj127 [DOI] [PubMed] [Google Scholar]
  33. Ohara‐Imaizumi, M. , Nishiwaki, C. , Kikuta, T. , Nagai, S. , Nakamichi, Y. , & Nagamatsu, S. (2004). TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta‐cells: Different behaviour of granule motion between normal and Goto‐Kakizaki diabetic rat beta‐cells. The Biochemical Journal, 381(Pt 1), 13–18. 10.1042/BJ20040434 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Papadopulos, A. , Gomez, G. A. , Martin, S. , Jackson, J. , Gormal, R. S. , Keating, D. J. , Yap, A. S. , & Meunier, F. A. (2015). Activity‐driven relaxation of the cortical actomyosin II network synchronizes Munc18‐1‐dependent neurosecretory vesicle docking. Nature Communications, 6, 6297. 10.1038/ncomms7297 [DOI] [PubMed] [Google Scholar]
  35. Richmond, J. E. , Weimer, R. M. , & Jorgensen, E. M. (2001). An open form of syntaxin bypasses the requirement for UNC‐13 in vesicle priming. Nature, 412(6844), 338–341. 10.1038/35085583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rudolf, R. , Salm, T. , Rustom, A. , & Gerdes, H. H. (2001). Dynamics of immature secretory granules: Role of cytoskeletal elements during transport, cortical restriction, and F‐actin‐dependent tethering. Molecular Biology of the Cell, 12(5), 1353–1365. 10.1091/mbc.12.5.1353 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schuh, M. (2011). An actin‐dependent mechanism for long‐range vesicle transport. Nature Cell Biology, 13(12), 1431–1436. 10.1038/ncb2353 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Seino, S. , Shibasaki, T. , & Minami, K. (2011). Dynamics of insulin secretion and the clinical implications for obesity and diabetes. The Journal of Clinical Investigation, 121(6), 2118–2125. 10.1172/JCI45680 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shibasaki, T. , Takahashi, H. , Miki, T. , Sunaga, Y. , Matsumura, K. , Yamanaka, M. , Zhang, C. , Tamamoto, A. , Satoh, T. , Miyazaki, J. I. , & Seino, S. (2007). Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP. Proceedings of the National Academy of Sciences of the United States of America, 104(49), 19333–19338. 10.1073/pnas.0707054104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takahashi, N. , Sawada, W. , Noguchi, J. , Watanabe, S. , Ucar, H. , Hayashi‐Takagi, A. , Yagishita, S. , Ohno, M. , Tokumaru, H. , & Kasai, H. (2015). Two‐photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and beta cells. Nature Communications, 6, 8531. 10.1038/ncomms9531 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Torii, S. , Takeuchi, T. , Nagamatsu, S. , & Izumi, T. (2004). Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a. The Journal of Biological Chemistry, 279(21), 22532–22538. 10.1074/jbc.M400600200 [DOI] [PubMed] [Google Scholar]
  42. Torii, S. , Zhao, S. , Yi, Z. , Takeuchi, T. , & Izumi, T. (2002). Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a. Molecular and Cellular Biology, 22(15), 5518–5526. 10.1128/mcb.22.15.5518-5526.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Van Gele, M. , Dynoodt, P. , & Lambert, J. (2009). Griscelli syndrome: A model system to study vesicular trafficking. Pigment Cell & Melanoma Research, 22(3), 268–282. 10.1111/j.1755-148X.2009.00558.x [DOI] [PubMed] [Google Scholar]
  44. Verhage, M. , & Sorensen, J. B. (2008). Vesicle docking in regulated exocytosis. Traffic, 9(9), 1414–1424. 10.1111/j.1600-0854.2008.00759.x [DOI] [PubMed] [Google Scholar]
  45. Wang, H. , Ishizaki, R. , Kobayashi, E. , Fujiwara, T. , Akagawa, K. , & Izumi, T. (2011). Loss of granuphilin and loss of syntaxin‐1A cause differential effects on insulin granule docking and fusion. The Journal of Biological Chemistry, 286(37), 32244–32250. 10.1074/jbc.M111.268631 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wang, H. , Mizuno, K. , Takahashi, N. , Kobayashi, E. , Shirakawa, J. , Terauchi, Y. , Kasai, H. , Okunishi, K. , & Izumi, T. (2020). Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane. Diabetes, 69(12), 2655–2666. 10.2337/db20-0069 [DOI] [PubMed] [Google Scholar]
  47. Wu, B. , & Guo, W. (2015). The exocyst at a glance. Journal of Cell Science, 128(16), 2957–2964. 10.1242/jcs.156398 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Xie, L. , Zhu, D. , & Gaisano, H. Y. (2012). Role of mammalian homologue of Caenorhabditis elegans unc‐13‐1 (Munc13‐1) in the recruitment of newcomer insulin granules in both first and second phases of glucose‐stimulated insulin secretion in mouse islets. Diabetologia, 55(10), 2693–2702. 10.1007/s00125-012-2640-z [DOI] [PubMed] [Google Scholar]
  49. Yi, Z. , Yokota, H. , Torii, S. , Aoki, T. , Hosaka, M. , Zhao, S. , Takata, K. , Takeuchi, T. , & Izumi, T. (2002). The Rab27a/granuphilin complex regulates the exocytosis of insulin‐containing dense‐core granules. Molecular and Cellular Biology, 22(6), 1858–1867. 10.1128/mcb.22.6.1858-1867.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhao, K. , Matsunaga, K. , Mizuno, K. , Wang, H. , Okunishi, K. , & Izumi, T. (2023). Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis. eLife, 12, e82821. 10.7554/eLife.82821 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genes to Cells are provided here courtesy of Wiley

RESOURCES