Page 1 of 55 Diabetes
Melanophilin accelerates insulin granule fusion without predocking to the plasma
membrane
Hao Wang,1 Kouichi Mizuno,1 Noriko Takahashi,2 Eri Kobayashi,1 Jun Shirakawa,3 Yasuo
Terauchi,3 Haruo Kasai,4 Katsuhide Okunishi,1 and Tetsuro Izumi1*
1Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine,
Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, 371-
8512, Japan, 2Department of Physiology, Kitasato University School of Medicine, Sagamihara,
Kanagawa, 252-0373, Japan, 3Department of Endocrinology and Metabolism, Graduate School
of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan, 4Laboratory
of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of
Medicine, The University of Tokyo, Tokyo, 113-0033, Japan
A short running title: A fast mode of undocked granule exocytosis
*Correspondence: [email protected]
Address: 3-39-15 Showa-machi, Maebashi, Gunma 371-8512
Tel: +81-27-220-8856
The word count: 4121
The number of figures: 6
1
Diabetes Publish Ahead of Print, published online September 29, 2020 Diabetes Page 2 of 55
Abstract
Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated leaden mouse and melanophilin-downregulated
human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific
reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-
2+ induced [Ca ]i rise, melanophilin mediates this type of fusion by dissociating granules from
myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open
form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown
mechanism to support sustainable exocytosis by which granules are recruited from the cell
interior and fuse promptly without stable predocking to the plasma membrane.
2 Page 3 of 55 Diabetes
Introduction
Professional secretory cells store bioactive molecules in vesicles in advance and release them in
response to extracellular stimuli by promoting fusion of vesicle membranes to the plasma
membrane. In such regulated exocytic pathways, secretory granules carrying proteins as cargo
must be regenerated at the Golgi apparatus after releasing their contents, in contrast that synaptic
vesicles containing low-molecular-weight substances can recycle within the presynaptic
terminal. Therefore, newly generated granules must cross a peripheral microfilament web,
referred to as the actin cortex, before approaching the plasma membrane. However, the
molecular mechanism by which granules link to the F-actin network and are processed towards
exocytosis remains poorly understood. This process that accumulates granules in the cell
periphery is thought to form a reserve pool to sustain regulated secretion after depletion of a
readily releasable pool beneath the plasma membrane. Its disturbance could impair the capacity
of secretory cells to cope with external changes and stresses and cause diseases such as type 2
diabetes.
We have recently shown that exophilin-8 (also known as MyRIP and Slac2-c), one of
the Rab27 effectors that play versatile roles in regulated secretory pathways (1), captures
granules within the actin cortex via indirect interaction with myosin-VIIa through binding to
RIM-BP2, and that this exophilin-8-RIM-BP2-myosin-VIIa complex formation is critical for
peripheral accumulation and efficient exocytosis of insulin granules (2). However, another motor
protein on actin filaments, myosin-Va, has also been suggested to function as a carrier to capture
and/or transport granules to the vicinity of the plasma membrane (3-6), although the molecular
mechanism by which myosin-Va functions in granule exocytosis remains unknown. Myosin-Va
does not interact with exophilin-8, but binds another Rab27 effector, melanophilin (also known
3 Diabetes Page 4 of 55
as exophilin-3 and Slac2-a), in pancreatic β cells (2). Melanophilin retains melanosomes in the periphery of skin melanocytes by directly interacting with both Rab27a on melanosomes and myosin-Va on cortical actin filaments, which makes melanosomes capable of being transferred to neighboring keratinocytes (7-10). Its functional loss leads to clustering of melanosomes near the perikaryotic regions and causes hypopigmentation in both leaden mice and human Griscelli syndrome patients (11,12). However, other overt abnormalities have not been reported. The present study demonstrates in vivo function of melanophilin in insulin granule exocytosis that bypasses stable predocking to the plasma membrane.
4 Page 5 of 55 Diabetes
Research Designs and Methods
Mice and phenotypic characterization
Leaden (C57J/L) and C57BR/cdJ mice were purchased from the Jackson Laboratory. To
minimize potential effects of spontaneous mutations occurring after separation of these inbred
strains, C57L/J were crossed with C57BR/cdJ and resultant heterozygous mice were intercrossed
to generate Mlphln/Mlphln mice, which were used in the current study. Animal experiments were
performed according to the rules and regulations of the Animal Care and Experimentation
Committees of Gunma University and the University of Tokyo. Only male mice and their tissues
and cells were phenotypically characterized in this study. Blood glucose levels were determined
by a glucose oxidase method using Glutest Pro GT-1660 (Sanwa Kagaku Kenkyujyo). Insulin
was measured by an AlphaLISA insulin kit (PerkinElmer). Pancreatic islet isolation, perifusion
secretion assays, and morphometric electron microscopic analysis of granule distribution were
performed as described previously (13,14).
Antibodies and Immunoprocedures
The sources of antibodies and their concentrations used are listed in Supplementary Table 1.
Cells lysate proteins separated by gel electrophoresis were transferred onto an Immobilon-P
membrane (Millipore), and were visualized by means of enhanced chemiluminescence (GE
Healthcare Biosciences). Immunoprecipitation was performed at 4ºC by incubation with primary
antibody overnight followed by the addition of protein G-agarose beads (GE Healthcare
Bioscience) for 1 h, or by direct incubation with anti-HA affinity matrix beads (Roche
Diagnostics) or anti-FLAG affinity gel (Sigma-Aldrich) for 1 h. For immunofluorescence,
primary β cells were fixed by 4% paraformaldehyde for 30 min at room temperature, and were
5 Diabetes Page 6 of 55
rehydrated with PBS for 5 min followed by PBS plus 0.1% Triton X-100 for 30 min. The cells incubated with primary antibody overnight at 4ºC followed by Alexa Fluor 488- or 568- conjugated secondary antibody for 1 h at room temperature were observed by a confocal laser scanning microscope. Each image is representative of at least three independent experiments.
DNA and RNA manipulation
Mouse melanophilin and syntaxin-4 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy, and were verified by
DNA sequencing. The sequences of the primers used were listed in Supplementary Table 2.
These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG (Invitrogen), pmCherry-C1, pEGFP-C1 (Clontech), pMAL-cR1 (New England Biolabs), pGEX4T-1 (GE Healthcare
Bioscience), or pCAG with a One-STrEP-Flag (OSF) tag as described previously (2,13).
Neuropeptide Y (NPY)-mCherry cDNA was generated by subcloning a mCherry cDNA into the pNPY-Venus-N1 vector. To generate recombinant adenoviruses, they were inserted into pENTR-
3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen).
To express exogenous protein, HEK293A cells were transfected with the plasmids using
Lipofectamine 2000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.
Total internal reflection fluorescence (TIRF) microscopy
Human islets (Supplementary Human Islet Checklist) were provided by the Alberta Diabetes
Institute IsletCore of the University of Alberta under full ethical clearance (Yokohama City
University Ethics Board, B171100025 and Human Tissue MTA from the University of Alberta,
UA17-DSA-64). Mouse and human islets were dissociated into monolayer cells by incubation
6 Page 7 of 55 Diabetes
with trypsin-EDTA solution, and were cultured on poly-L-lysine-coated 35-mm glass base dishes
for 2 days. The cells were infected with adenovirus encoding preproinsulin-enhanced green
fluorescent protein (Insulin-EGFP) or NPY-mCherry, and was further cultured for 2 days. TIRF
microscopy (the penetration depth of the evanescent field: 100 nm) was performed as described
previously (15,16). The cells were preincubated for 30 min in 2.8 mM glucose-containing Krebs-
Ringer bicarbonate (KRB) buffer at 37ºC, and were exposed to 25 mM glucose stimulation for
20 min. Images were acquired at 103 ms intervals. Fusion events with a flash were manually
selected and assigned to one of three types: residents, which are visible over 10 s before fusion,
visitors, which have become visible within 10 s before fusion, and passengers, which are not
visible before fusion.
Silencing of melanophilin in human pancreatic islet cells
Human pancreatic islet cells suspended in 1 × 105 cells/200 μl were transfected with 50 nM
control On-Target plus non-targeting pool siRNA or SMARTpool siRNA against human
melanophilin (79083; GE Dharmacon) using Lipofectamine RNAiMAX reagent (Invitrogen).
After plated on glass base dish for 72 h, control and melanphilin siRNA-treated cells were
infected with 10 MOI of mCherry-tagged, nontargeting (ACTACCGTTGTTATAGGTG) and
human melanophilin targeting (GCGTTGAAGGGCAAGATTA) shRNA adenoviruses,
respectively. Cells intended for TIRF microscopy were coinfected with adenoviruses encoding
shRNA and Insulin-EGFP, wherein infected cells were determined by mCherry and EGFP
expression.
Two-photon excitation imaging
7 Diabetes Page 8 of 55
Two-photon extracellular polar tracer imaging of exocytic events in islets was performed as described previously (17). The external bathing solution contained 10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 2.8 mM glucose, and 0.7 mM sulforhodamine B. Exocytic events triggered with 20 mM glucose were counted in a region of interest with an area of 3254-4992 μm2, and normalized to an area of 800 μm2. The number of sequential exocytic events was quantified as described previously (18).
Statistical analysis
All quantitative data were expressed as the mean ± SEM. The P values were calculated using
Student’s t-test or a one-way ANOVA with a Dunnett multiple-comparison test.
The data and resource availability statements
The datasets and all noncommercially available resources generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
8 Page 9 of 55 Diabetes
Results
Melanophilin mutation causes a specific defect in undocked insulin granule exocytosis
Because Rab27a is localized on insulin granules and is involved in their exocytosis (19,20),
genetic mutation of any Rab27 effector could potentially cause a defect in insulin secretion. In
fact, leaden mice with a natural melanophilin mutation showed glucose intolerance compared
with their control mice, although body weight and blood glucose levels in a fasting state or after
an insulin load did not differ (Fig. 1A). Melanophilin was expressed in wild-type pancreatic
islets (Fig. 1B). We were unable to examine the intracellular distribution of endogenous
melanophilin due to lack of an antibody sufficiently durable for immunostaining. However, HA-
tagged melanophilin expressed at an endogenous level in monolayer pancreatic β cells was
observed to colocalize with insulin granules, especially those at the cell periphery (Fig. 1C,D).
Although melanophilin is known to interact with actin motor protein, myosin-Va, and/or actin
itself (7-10,21), its presence or absence did not affect insulin granule accumulation in the cell
corners, where F-actin and myosin-Va were enriched (Supplementary Fig. 1). Electron
microscopic analyses also revealed no significant changes in the number, density, or distribution
of granules, including the number of docked granules having centers residing within 200 nm of
the plasma membrane in leaden β cells (Supplementary Fig. 2). Perifusion assays of leaden islets
revealed a significant decrease in insulin secretion in response to glucose or a stronger stimulus,
glucose plus forskolin (Fig. 1E).
Confirming that the majority of monolayer cells derived from wild-type and leaden
pancreatic islets are β cells by insulin immunostaining (90.1% and 89.7%, respectively, n = 3
each), we compared fusion profiles in living cells under TIRF microscopy that visualizes Insulin-
EGFP-labeled granules just beneath the plasma membrane. Fused granules were categorized into
9 Diabetes Page 10 of 55
three types as described previously (15,16): residents, granules visible before stimulation;
visitors, granules visualized during stimulation; and passengers, granules invisible before fusion.
Despite the different prefusion behaviors, the average peak fluorescence intensity during fusion
was similar among the three types (15) (see also Fig. 2D for the case of human β cells),
suggesting that they all represent single granule exocytosis. The total number of fusion events,
especially that in the late phase of glucose stimulation, was markedly decreased in leaden β cells
(Fig. 2A-C), consistent with findings from the perifusion analysis (Fig. 1E). Remarkably, only
the passenger type was decreased by half. Although this type of exocytosis without stable
predocking to the plasma membrane has consistently been observed in rodent β cells expressing
either Insulin-EGFP (15,22) or Insulin-Venus (23) and in human β cells expressing NPY-EGFP
(24-26), its presence has recently been disputed in human β cells expressing NPY-mCherry (27).
However, because the passenger type is visible only in one frame (103 ms) as a flash by granule
neutralization during the fusion, it might be difficult to find if granules are visualized by pH-
insensitive mCherry (28). In fact, in contrast to the granules labeled by Insulin-EGFP, those
labeled by NPY-mCherry showed no fluorescence intensity peak even during the resident type of
fusion in mouse β cells (Video 1, Supplementary Fig. 3A). Accordingly, they revealed almost no
passenger type but similar numbers of other types (Supplementary Fig. 3B). Thus, the
discrepancy should not have arisen from a difference in animal species of β cells, but from a
difference in fluorescent proteins used to visualize granules. We confirmed in human β cells that
the passenger type of exocytosis occurs at a frequency of ~40% during 20-min glucose
stimulation, though the visitor type of exocytosis is hardly seen, and that knockdown of human
melanophilin selectively decreases passenger type exocytic events (Fig. 2D and E; Videos 2 and
3; Supplementary Fig. 4). We then expressed mCherry-melanophilin to label passengers in β
10 Page 11 of 55 Diabetes
cells. However, mCherry-melanophilin showed too weak fluorescence to monitor granule
exocytosis if expressed at an endogenous level, and aberrant localization along F-actin if
overexpressed (Supplementary Fig. 5A). Nevertheless, immunostaining with anti-RFP antibody
in cells fixed after 20-min glucose stimulation revealed that mCherry-melanophilin expressed at
an endogenous level exists at the site of passenger exocytosis especially where the exocytosis
has occurred at later time points and thus the fused granules and associated proteins likely
remain at the plasma membrane (Supplementary Fig. 5B). Although EGFP-melanophilin
exhibited stronger fluorescence and colocalized with NPY-mCherry (Supplementary Fig. 6A),
this set of fluorescent proteins cannot be used to detect passenger exocytosis as described.
Further, EGFP-melanophilin colocalized with coexpressed Kusabira-Orange 1 (KuO)-
granuphilin, another Rab27 effector mediating resident type of exocytosis (29). We confirmed
that HA-melanophilin colocalizes with endogenous granuphilin under confocal microscopy
(Supplementary Fig. 6B). These findings indicate that a single granule can carry different Rab27
effectors simultaneously, and that melanophilin is not specifically locates on granules showing
passenger exocytosis.
The passenger type might correspond to sequential exocytosis wherein granules fuse
selectively with other granules that have already fused with the plasma membrane, although the
frequency of sequential exocytosis is minor (2-3%) in mouse pancreatic islets (18), compared
with that of passenger exocytosis found in mouse monolayer β cells (~40%, Fig. 2C). To exclude
this possibility, we performed two-photon excitation imaging of wild-type and leaden mouse
islets. Although the number of exocytic events during the first 5 min of glucose stimulation was
not different, that during the 5-15 min stimulation was markedly reduced in leaden islets (Fig.
3A and B). This time-dependent difference may correspond to current and previous TIRF
11 Diabetes Page 12 of 55
microscopic findings (Fig. 2A) (15,22,23) that the relative frequency of the passenger type of exocytosis increases in the late phase of glucose-stimulated insulin secretion (GSIS). However, the frequency of sequential exocytosis did not differ between wild-type and leaden islets (3.3 ±
0.6% vs. 3.1 ± 1.6%, Fig. 3C), indicating that melanophilin is not involved in sequential exocytosis.
Melanophilin interacts with syntaxin-4
To identify the molecular mechanism by which melanophilin regulates granule exocytosis, we first compared the expression levels of proteins known to interact with melanophilin or to function in insulin granule exocytosis between wild-type and leaden islets. We found that syntaxin-4, a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), was expressed at significantly lower levels in leaden islets (Supplementary
Fig. 7). Because protein expression levels often decline with loss of an interacting protein, and because syntaxin-4 is known to function in insulin granule exocytosis (25,30), we explored the possibility that melanophilin interacts with syntaxin-4. Melanophilin exogenously expressed in the pancreatic β-cell line MIN6 coprecipitated syntaxin-4 as well as previously known melanophilin-interacting proteins, such as Rab27a, myosin-Va, β-actin, and EB1 (Fig. 4A). In contrast, melanophilin did not interact with syntaxin-1a, -2, or -3. We confirmed that melanophilin forms an endogenous complex with syntaxin-4 in pancreatic islets (Fig. 4B).
This newly identified interaction might be mediated indirectly via actin, because both proteins have been reported to bind actin directly (21,30). To investigate this possibility, we first determined the interacting regions in these two proteins. The cytoplasmic region of syntaxin-4 consists of the N-terminal Habc domain and the C-terminal H3 domain containing a SNARE
12 Page 13 of 55 Diabetes
motif. When each domain without the transmembrane domain was coexpressed with
melanophilin in HEK293A cells, only the H3 domain formed a complex (Fig. 4C). On the other
hand, melanophilin comprises the N-terminal Rab27a binding domain (RBD), the central
myosin-Va binding domain (MBD), and the C-terminal actin binding domain (ABD). We found
that the N-terminal protein covering the RBD and the MBD interacts with syntaxin-4, whereas
the C-terminal protein covering the MBD and the ABD does not (Fig. 4D, left). Furthermore,
although the MBD domain only (147-400 amino acid residues) or that with additional N-terminal
residues (126-400) could not bind syntaxin-4, the MBD with further N-terminal residues (116-
400) was able to bind it (Fig. 4D, right). Because melanophilin binds actin through the ABD
(401-590 residues) (21), and because syntaxin-4 interacts with actin through the Hab domains
(39-112 residues) (31), the regions responsible for the interaction between melanophilin and
syntaxin-4 are completely different from the actin-binding region of each protein. Therefore, it is
unlikely that the melanophilin-syntaxin-4 complex is indirectly mediated through the actin
binding. We confirmed that the N-terminal melanophilin (1-400 residues) bound syntaxin-4
without involvement of other specific proteins in HEK293A cells (Fig. 4E). Furthermore, the
interaction between melanophilin (1-146 residues) and syntaxin-4 (1-273 residues) was observed
both in MIN6 cell extracts (Fig. 4F) and between bacterially expressed, purified proteins (Fig.
4G, Supplementary Fig. 8).
These findings indicate that residues 116-125 of melanophilin are crucial for the
interaction with syntaxin-4. In fact, the N-terminal 1-130 residues, but not the 1-120 residues,
could efficiently bind syntaxin-4 (Fig. 4H). The generation of point mutations around this region
revealed that the Y122A/H124A/K130A triple mutant exhibits a greatly reduced syntaxin-4-
binding activity (Fig. 4I). The 117-122 residues, SLEWYY, of melanophilin correspond to the
13 Diabetes Page 14 of 55
SGAWFF structural element of rabphilin that interacts with a deep pocket in Rab3a in the crystal structure (32). Furthermore, the Y121 of melanophilin has been shown to make a hydrogen bond with the R90 carbonyl group of Rab27b (33). Therefore, the triple mutant might also affect the interaction with Rab27a. However, the mutant had a Rab27a-binding activity comparable to that of full-length melanophilin (Fig. 4I), which is consistent with previous findings that either the
W120A/Y121A mutation (8) or the broader deletion of 111-145 residues (34) of melanophilin does not affect the interaction with Rab27a. Thus, the Y122A/H124A/K130A mutant specifically loses binding activity to syntaxin-4.
Melanophilin promotes insulin exocytosis via interactions with Rab27a, myosin-Va, and syntaxin-4
To examine whether the interaction with syntaxin-4 is important for melanophilin’s promotion of insulin granule exocytosis, we first confirmed that the Y122A/H124A/130A mutant loses binding activity to endogenous syntaxin-4, but not to Rab27a or myosin-Va, in MIN6 cells (Fig.
5A). In contrast, the E14A and D378A/E380A/E381A/E382A/A467P mutants selectively disrupted the interaction with Rab27a and myosin-Va, respectively, as previously reported
(8,21,35), but not that with syntaxin-4. Immunostaining revealed that the Y122A/H124A/130A mutant expressed in leaden β cells does not colocalize with syntaxin-4 along the plasma membrane, but is only distributed to the F-actin-rich cell corners (Fig. 5B). In contrast, the E14A mutant did not colocalize with the granule-resident Rab27a at all, whereas the
D378A/E380A/E381A/E382A/A467P mutant did not colocalize with myosin-Va accumulated at the cell corners, but located only along the plasma membrane. We then performed rescue experiments by introducing wild-type or mutant melanophilin in leaden islets to match the level
14 Page 15 of 55 Diabetes
of endogenous melanophilin in wild-type islets (Fig. 5C). The wild-type melanophilin tended to
increase the expression level of syntaxin-4 in leaden islets (Fig. 5C, upper right), and restored
GSIS to the level found in wild-type islets expressing the control LacZ protein (Fig. 5D). In
contrast, the E14A mutant did not restore insulin secretion at all, confirming the importance of
melanophilin’s association with the granule membrane via Rab27a. Neither the
Y122A/H124A/K130A nor the D378A/E380A/E381A/E382A/A467P mutant exhibited an
enhancement, suggesting that the interactions with syntaxin-4 and myosin-Va are also important.
To obtain further specific evidence, we performed rescue experiments in monolayer β cells under
TIRF microscopy (Fig. 5E). Leaden β cells expressing wild-type melanophilin showed a specific
increase in the number of passenger type exocytic events in response to glucose stimulation. In
contrast, those expressing the Y122A/H124A/K130A or the
D378A/E380A/E381A/E382A/A467P mutant did not exhibit such an increase.
Melanophilin shows Ca2+-dependent interactions with the open form of syntaxin-4 and
myosin-Va in cells
To explore the mechanism underlying melanophilin’s acceleration of exocytosis without a
significant presence beneath the plasma membrane, we investigated the mode of interaction
between melanophilin and syntaxin-4. Syntaxin members are thought to exist in an equilibrium
between the open and closed conformations, and only the open form is capable of forming a
trans-SNARE complex with other SNARE proteins to mediate a fusion reaction (36,37). The
syntaxin-4 mutant L173A/E174A corresponds to the syntaxin-1a mutant L165A/E166A that
adopts a constitutively open conformation (38). We found that exogenously expressed
melanophilin interacts with wild-type and L173A/E174A syntaxin-4 similarly in HEK293A cells
15 Diabetes Page 16 of 55
(Fig. 6A), suggesting that it preferentially binds the fusion-competent, open form. This binding mode could account for the passenger type of instant fusion seen after granules approach the plasma membrane. Importantly, the interaction of melanophilin with syntaxin-4, and its SNARE partners, VAMP2 and SNAP25, was induced by glucose stimulation, and this induction disappeared in the simultaneous presence of the Ca2+-chelator EGTA, in MIN6 cells (Fig. 6B and
C). This finding suggests that the complex forms only after the stimulus-induced, intracellular
Ca2+ increase, and is consistent with its role in mediating the passenger type of exocytosis wherein granules are recruited to the plasma membrane only after stimulation. This stimulus- dependent binding was similarly observed for the L173A/E174A mutant (Fig. 6D), suggesting that Ca2+ does not directly alter the conformation of syntaxin-4, but allows melanophilin-positive granules to reach syntaxin-4 on the plasma membrane by breaking down the F-actin network.
Consistent with this idea, melanophilin promptly but transiently dissociated from myosin-Va and actin upon glucose stimulation in a Ca2+-dependent manner (Fig. 6E and F).
16 Page 17 of 55 Diabetes
Discussion
Under TIRF microscopy visualizing secretory granules using pH-sensitive fluorescent protein,
we clearly showed that granules exhibit heterogeneous prefusion behaviors in both mouse and
human pancreatic β cells. The majority of the resident type of exocytosis likely corresponds to
the mode of fusion preceded by stable docking to the plasma membrane (29), and several
molecules, such as granuphilin and RIM2, have been shown to tether and/or dock granules to the
plasma membrane (14,39). In contrast, the molecular mechanism of the latter passenger
(undocked) type of exocytosis has not been explored well. The previous findings that syntaxin-4,
Munc18-3, and synaptotagmin-7 are involved in both types of exocytosis (24-26) indicate that
the passenger type also involves such general exocytic machinery components and represents a
real exocytic phenomenon. However, the molecular mechanism unique to this type has still been
enigmatic. Given that this type of exocytosis becomes dominant in the late phase of GSIS, it
should help sustain granule exocytosis in a prolonged time. Rab27 effectors, such as
melanophilin and exophilin-8, are good candidates to form such a reserve pool of granules,
because they can link Rab27 on the granule membrane and myosin motors within the cell
peripheral actin network. We have recently shown that silencing of each component of the
exophilin-8-RIM-BP2-myosin-VIIa complex markedly decreases the peripheral accumulation
and exocytosis of insulin granules (2). In contrast, loss of melanophilin (present study) or
silencing of melanophilin-interacting myosin-Va (2) decreases GSIS, but does not affect the
granule accumulation at the cell periphery. These findings suggest that exophilin-8 first acts to
capture granules within a relatively broad area of the actin cortex (2,40), whereas melanophilin
and myosin-Va then function to promote granule exocytosis beneath the plasma membrane.
Indeed, melanophilin mediates the passenger type of exocytosis via its interactions with myosin-
17 Diabetes Page 18 of 55
Va and syntaxin-4a. Myosin-Va unlikely functions as an active motor for granule movement, because granules showing this type of exocytosis must pass the evanescent field (100~200 nm) per 100 ms (one frame of TIRF microscopy) before fusion, the velocity of which is above its motor speed. A faster kinesin motor may work in their recruitment to the plasma membrane. In any case, those granules should not have located in a deep cell interior, but just above the evanescent field, still close to the plasma membrane, before they fuse in response to external stimulation.
Because granules showing the passenger type of exocytosis are stimulus-dependently recruited from the cell interior and mediate instant fusion without pausing beneath the plasma membrane, they must promptly assemble the fusion machinery after stimulation (41,42). The stimulus-dependent dissociation of melanophilin from myosin-Va would allow granules to be released from the F-actin network and to associate with fusion machinery, syntaxin-4, on the plasma membrane. Further, the specific interaction of melanophilin with the fusion-competent, open form of syntaxin-4 and its SNARE partners in cells after stimulation, should enable granules to fuse instantly with the plasma membrane. In this context, it is interesting that syntaxin-4 interacts with the Ca2+-activated F-actin-serving protein, gelsolin, and that the complex-dissociating action of secretagogues can induce the open form of syntaxin-4 in MIN6 cells (43), which might facilitate the complex formation between melanophilin and syntaxin-4 at actin filament tips adjacent to the plasma membrane (44). Alternatively, SNAREs in native plasma membranes are constitutively active even if not engaged in fusion events, as previously shown (45). Although the interaction of melanophilin with syntaxin-4 is reminiscent of that of another Rab27 effector, granuphilin, with syntaxins-1a, -2 and -3 (13,14,46). However, granuphilin specifically interacts with the fusion-incompetent, closed-form of syntaxin-1a in the
18 Page 19 of 55 Diabetes
absence of Ca2+ (47), and exclusively mediates exocytosis of fusion-reluctant, stably docked
granules (14,15,29). Because melanophilin and granuphilin coexist on almost all the granules in
β cells, the heterogeneous modes of granule exocytosis observed in living cells should not be
determined by the existence of specific Rab27 effector per se, but may reflect the existence of
distinct modes of interaction between specific tethering factors (Rab27 effectors) and fusion
machinery (syntaxins).
In summary, melanophilin links granules within the peripheral actin network via
interactions with Rab27a and myosin-Va, dissociates them from it by stimulus-induced F-actin
dissolution, and then interacts with the open form of syntaxin-4 to induce immediate fusion.
Although loss of melanophilin does not abolish the passenger type of exocytosis, that which
remains in leaden β cells may be mediated by other Rab27 effectors, such as exophilin-7 (16)
and exophilin-8 (2,40), or may correspond to sequential exocytosis. Parallel and/or redundant
exocytic pathways involving predocked and undocked granules would guarantee robustness to
the secretory process that produces many indispensable components, including insulin.
19 Diabetes Page 20 of 55
Acknowledgments. We thank Prof. P. E. MacDonald (Alberta Diabetes Institute IsletCore) for supplying human pancreatic islets, Prof. A. Miyawaki (RIKEN Brain Science Institute) for providing the pNPY-Venus-N1 vector, H. Kobayashi (Gunma University) for providing guinea pig anti-porcine insulin serum, and the members of Laboratory of Molecular Endocrinology and
Metabolism, especially Dr. K. Matsunaga for useful suggestion and discussion, T. Nara and T.
Ushigome for their colony management of mice, and S. Shigoka and J. Toshima for assistance in preparing the manuscript.
Funding. This work was supported by JSPS KAKENHI grants JP26670133, JP14F04104, and
JP16K15211 to T.I., JP18K14647 and JP20K15742 to H.W., and JST-CREST grant JPMJCR1652 to H.K. It was also supported by grants from Uehara Memorial Foundation, Kobayashi
International Scholarship Foundation, Novartis Research Grants, Pfizer Academic Contributions,
Astellas Research Support, MSD Scholarship Donation, and Sanofi Scholarship Donation to T.I.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. H.W. and K.M. performed experiments and analyzed data, N.T. performed two-photon excitation microscopy and analyzed data, E.K. performed electron microscopy, J.S. and Y.T. provided experimental reagents, H.K. and K.O. analyzed data, and T.I. designed experiments and wrote the paper. T.I. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
20 Page 21 of 55 Diabetes
Prior Presentation. Parts of this study were presented in abstract form at the EASD Annual
Meeting 2019, Barcelona, Spain, 16–20 September 2019.
21 Diabetes Page 22 of 55
References
1. Izumi T, Gomi H, Kasai K, Mizutani S, Torii S. The roles of Rab27 and its effectors in the regulated secretory pathways. Cell Struct Funct 2003;28:465-474 2. Fan F, Matsunaga K, Wang H, Ishizaki R, Kobayashi E, Kiyonari H, Mukumoto, Y., Okunishi, K., and Izumi, T. Exophilin-8 assembles secretory granules for exocytosis in the actin cortex via interaction with RIM-BP2 and myosin-VIIa. Elife 2017;6:e26174 3. Rudolf R, Kogel T, Kuznetsov SA, Salm T, Schlicker O, Hellwig A, Hammer JA 3rd, Gerdes HH. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells. J Cell Sci 2003;116:1339-1348 4. Varadi A, Tsuboi T, and Rutter GA. Myosin Va transports dense core secretory vesicles in pancreatic MIN6 Myosin β-cells. Mol Biol Cell 2005;16:2670-2680 5. Ivarsson R, Jing X, Waselle L, Regazzi R, Renstrom E. Myosin 5a controls insulin granule recruitment during late-phase secretion. Traffic 2005;6:1027-1035 6. Desnos C, Huet S, Fanget I, Chapuis C, Bottiger C, Racine V, Sibarita JB, Henry JP, Darchen F. Myosin Va mediates docking of secretory granules at the plasma membrane. J Neurosci 2007;27:10636-10645 7. Fukuda M, Kuroda TS, Mikoshiba K. Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J Biol Chem 2002;277:12432-12436 8. Nagashima K, Torii S, Yi Z, Igarashi M, Okamoto K, Takeuchi T, Izumi T. Melanophilin directly links Rab27a and myosin Va through its distinct coiled-coil regions. FEBS letters 2002;517:233-238 9. Strom M, Hume AN, Tarafder AK, Barkagianni E, Seabra MC. A family of Rab27- binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. J Biol Chem 2002;277:25423-25430 10. Wu XS, Rao K, Zhang H, Wang F, Sellers JR, Matesic LE, Copeland NG, Jenkins NA, and Hammer JA 3rd. Identification of an organelle receptor for myosin-Va. Nat Cell Biol 2002;4:271-278 11. Matesic LE, Yip R, Reuss AE, Swing DA, O'Sullivan TN, Fletcher CF, Copeland NG, Jenkins NA. Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defects observed in leaden mice. Proc Natl Acad Sci U S A 2001;98:10238-10243 12. Ménasché G, Ho CH, Sanal O, Feldmann J, Tezcan I, Ersoy F, Houdusse A, Fischer A, de Saint Basile G. Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). J Clin Invest 2003;112:450-456 13. Wang H, Ishizaki R, Kobayashi E, Fujiwara T, Akagawa K, Izumi T. Loss of granuphilin and loss of syntaxin-1A cause differential effects on insulin granule docking and fusion. J Biol Chem 2011;286:32244-32250 14. Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T. Granuphilin molecularly docks insulin granules to the fusion machinery. J Cell Biol 2005;171:99-109 15. Kasai K, Fujita T, Gomi H, Izumi T. Docking is not a prerequisite but a temporal constraint for fusion of secretory granules. Traffic 2008;9:1191-203 16. Wang H, Ishizaki R, Xu J, Kasai K, Kobayashi E, Gomi H, Izumi T. The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma
22 Page 23 of 55 Diabetes
membrane. Mol Biol Cell 2013;24:319-330 17. Takahashi N, Kishimoto T, Nemoto T, Kadowaki T, Kasai H. Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet. Science 2002;297:1349-1352 18. Takahashi N, Hatakeyama H, Okado H, Miwa A, Kishimoto T, Kojima T, Abe T, Kasai H. Sequential exocytosis of insulin granules is associated with redistribution of SNAP25. J Cell Biol 2004;165:255-262 19. Yi Z, Yokota H, Torii S, Aoki T, Hosaka M, Zhao S, Takata K, Takeuchi T, Izumi T. The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules. Mol Cell Biol 2002;22:1858-1867 20. Kasai K, Ohara-Imaizumi M, Takahashi N, Mizutani S, Zhao S, Kikuta T, Kasai H, Nagamatsu S, Gomi H, Izumi T. Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation. J Clin Invest 2005;115:388-396 21. Kuroda TS, Ariga H, Fukuda M. The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes. Mol Cell Biol 2003;23:5245-5255 22. Ohara-Imaizumi M, Nishiwaki C, Kikuta T, Nagai S, Nakamichi Y, Nagamatsu S. TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat β-cells. Biochem J 2004;381:13-18 23. Shibasaki T, Takahashi H, Miki T, Sunaga Y, Matsumura K, Yamanaka M, Zhang C, Tamamoto A, Satoh T, Miyazaki J, Seino S. Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP. Proc Natl Acad Sci U S A 2007;104:19333-19338 24. Zhu D, Xie L, Karimian N, Liang T, Kang Y, Huang YC, Gaisano HY. Munc18c mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose stimulated insulin secretion in human pancreatic beta-cells. Mol Metab 2015;4:418-426 25. Xie L, Zhu D, Dolai S, Liang T, Qin T, Kang Y, Xie H, Huang YC, Gaisano HY. Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells. Diabetologia 2015;58:1250-1259 26. Dolai S, Xie L, Zhu D, Liang T, Qin T, Xie H, Kang Y, Chapman ER, Gaisano HY. Synaptotagmin-7 functions to replenish insulin granules for exocytosis in human islet β- cells. Diabetes 2016;65:1962-1976 27. Gandasi NR, Yin P, Omar-Hmeadi M, Ottosson Laakso E, Vikman P, Barg S. Glucose- dependent granule docking limits insulin secretion and is decreased in human type 2 diabetes. Cell Metab 2018;27:470-478 e4 28. Gandasi NR, Vesto K, Helou M, Yin P, Saras J, Barg S. Survey of red fluorescence proteins as markers for secretory granule exocytosis. PLoS One 2015;10:e0127801 29. Mizuno K, Fujita T, Gomi H, Izumi T. Granuphilin exclusively mediates functional granule docking to the plasma membrane. Sci Rep 2016;6:23909. 30. Spurlin BA, Thurmond DC. Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic β-cells. Mol Endocrinol 2006;20:183-193 31. Jewell JL, Luo W, Oh E, Wang Z, Thurmond DC. Filamentous actin regulates insulin exocytosis through direct interaction with Syntaxin 4. J Biol Chem 2008;283:10716- 10726 32. Ostermeier C, Brunger AT. Structural basis of Rab effector specificity: crystal structure
23 Diabetes Page 24 of 55
of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell 1999;96:363-374 33. Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2- a/melanophilin for the Rab27 GTPases. Structure 2008;16:1478-1490 34. Fukuda M. Synaptotagmin-like protein (Slp) homology domain 1 of Slac2- a/melanophilin is a critical determinant of GTP-dependent specific binding to Rab27A. J Biol Chem 2002;277:40118-40124 35. Hume AN, Tarafder AK, Ramalho JS, Sviderskaya EV, Seabra MC. A coiled-coil domain of melanophilin is essential for Myosin Va recruitment and melanosome transport in melanocytes. Mol Biol Cell 2006;17:4720-4735 36. Misura KM, Scheller RH, Weis WI. Three-dimensional structure of the neuronal-Sec1- syntaxin 1a complex. Nature 2000;404:355-362 37. Südhof TC, Rothman JE. Membrane fusion: grappling with SNARE and SM proteins. Science 2009;323:474-477 38. Dulubova I, Sugita S, Hill S, Hosaka M, Fernandez I, Südhof TC, Rizo J. A conformational switch in syntaxin during exocytosis: role of munc18. Embo J 1999;18:4372-4382 39. Yasuda T, Shibasaki T, Minami K, Takahashi H, Mizoguchi A, Uriu Y, Numata T, Mori Y, Miyazaki J, Miki T, Seino S. Rim2α determines docking and priming states in insulin granule exocytosis. Cell Metab 2010;12:117-129 40. Mizuno K, Ramalho JS, Izumi T. Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis. Mol Biol Cell 2011;22:1716-1726 41. Kasai H, Takahashi N, Tokumaru H. Distinct initial SNARE configurations underlying the diversity of exocytosis. Physiol Rev 2012;92:1915-1964 42. Takahashi N, Sawada W, Noguchi J, Watanabe S, Ucar H, Hayashi-Takagi A, Yagishita S, Ohno M, Tokumaru H, Kasai, H. Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells. Nat Commun 2015;6:8531 43. Kalwat MA, Wiseman DA, Luo W, Wang Z, Thurmond DC. Gelsolin associates with the N terminus of syntaxin 4 to regulate insulin granule exocytosis. Mol Endocrinol 2012;26;128-141 44. Tomas A, Yerman B, Min L, Pessin JE, Halban PA. Regulation of pancreatic β-cell insulin secretion by actin cytoskeleton remodeling: role of gelsolin and cooperation with the MAPK signaling pathway. J Cell Sci 2006;119,2156-2167 45 Lang T, Margittai M, Holzler H, Jahn R. SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs. J Cell Biol 2002;158:751-760 46 Torii S, Takeuchi T, Nagamatsu S, Izumi T. Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a. J Biol Chem 2004;279:22532-22538 47. Torii S, Zhao S, Yi Z, Takeuchi T, Izumi T. Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a. Mol Cell Biol 2002;22:5518- 5526
24 Page 25 of 55 Diabetes
Figure Legends
Figure 1. Leaden mice show glucose intolerance and impaired insulin secretion
(A) Body weight (left), blood glucose concentrations during an intraperitoneal glucose tolerance
test (1 g glucose/kg body weight; middle), and percentages of starting blood glucose
concentration during an intraperitoneal insulin tolerance test (0.75 U human insulin/kg body
weight; right). Each measurement was performed in age-matched (8- to 11-week-old), wild-type
(WT; open bars or circles) and leaden (closed bars or circles) mice (n = 11 each). (B) Protein
extracts (50 μg) from the indicated tissues from wild-type and leaden mice were analyzed by
immunoblotting with anti-melanophilin and anti-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antibodies. (C) Monolayer pancreatic β cells isolated from WT and leaden mice were
infected with adenoviruses encoding LacZ or HA-melanophilin. The protein level of HA-
melanophilin in leaden islets were adjusted to that of endogenous melanophilin in WT islets by
immunoblotting with anti-melanophilin antibody. (D) Under the condition in (C), monolayer
pancreatic β cells isolated from leaden mice were infected with adenovirus expressing HA-
melanophilin, and were coimmunostained with anti-HA and anti-insulin antibodies. Insets
represent higher magnification micrographs of a cell within the region outlined by frames. Bar, 5
μm. (E) Islets from age-matched (9- to 16-week-old), wild-type (open circles) or leaden mice
(closed circles) were stimulated by 16.7 mM glucose for 30 min (left, n = 7 each), or for 20 min
with pre- and post-incubation of 2.8 mM glucose buffer containing 10 μM forskolin for 15 min
(right, n = 5 each). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test.
Figure 2. Melanophilin deficiency decreases undocked granule exocytosis in mouse and
human β cells
25 Diabetes Page 26 of 55
(A, B) TIRF microscopic images were sampled every 103 ms in living wild-type (A) or leaden mouse islet cells (B) expressing Insulin-EGFP. All fusion events during 25 mM glucose stimulation for 20 min were manually counted in each cell (n = 32 cells from 10 mice each).
Time 0 indicates the initiation of stimulation. The histograms (left) show the average numbers of fusion events per 200 μm2 at 1-min intervals characterized as residents (black), visitors (gray), and passengers (white). White boxes in still cell images at a resting state (right) indicate the positions where the passenger type of exocytosis is observed during the stimulation, as examples. The yellow lines represent the outline of cells. Bars, 5 μm. (C) The numbers of fusion events from each type are summed during the early (from 1 to 7 min; left) and late (from 8 to 20 min; right) phases in wild-type (open bars) or leaden β cells (closed bars). (D) A monolayer of human pancreatic islet cells expressing Insulin-EGFP was stimulated by 25 mM glucose under
TIRF microscopy. An example of residents and passengers is shown. White boxes indicate the time of the beginning of fusion (left). The relative fluorescence intensity for each type was calculated in a 1 μm × 1 μm square around individual vesicles at each time point before and after fusion, and was normalized by that at 0.5 s after the peak at the same location (right; n = 5). Bar,
1 μm. (E) Melanophilin is downregulated in human islet cells (n = 13 cells from 4 donors), as shown in Supplementary Fig. 4. Under TIRF microscopy, the numbers of fusion events during
25 mM glucose stimulation for 20 min are summed in control (open bars) and melanophilin- downregulated (closed bars) human cells expressing Insulin-EGFP (upper), as in (C) for mouse cells. There is almost no visitor type of exocytosis in human cells. TIRF microscopic images of a cell coexpressing shRNA/mCherry and Insulin-EGFP are shown as an example (lower). Bars, 5
μm. *P < 0.05; Student’s t-test.
26 Page 27 of 55 Diabetes
Figure 3. The frequency of sequential exocytosis does not change in leaden islets
Islets isolated from wild-type (open circles, n = 8 from 5 mice) or leaden mice (closed circles, n
= 11 from 5 mice) were stimulated by 20 mM glucose. (A) Average number of exocytic events
detected in sulforhodamine B normalized per 800 μm2 per min. (B) Summed numbers of
exocytic events that occurred 0-300 s, 300-600 s, and 600-900 s after glucose stimulation. (C)
Frequency of sequential exocytosis during the 20 mM glucose stimulation (0-900 s). **P < 0.01,
***P < 0.001; Student’s t-test.
Figure 4. Melanophilin interacts with syntaxin-4
(A) MIN6 cells grown in 25 mM glucose-containing Dulbecco’s modified Eagle’s medium
supplemented with 15% were infected with adenovirus encoding either HA-melanophilin or
LacZ. The immunoprecipitate (IP) with anti-HA antibody, as well as 1/100 volume of the
original lysates (Input), were electrophoresed in 6%, 10%, and 12% polyacrylamide gels, and
were analyzed by immunoblotting with the indicated antibodies. (B) Total islet protein lysates
(300 μg) from wild-type mice were subjected to immunoprecipitation with anti-syntaxin-4
antibody or control IgG. The immunoprecipitates, as well as 1:12 of the original lysates, were
immunoblotted with anti-melanophilin and anti-syntaxin-4 antibodies. (C) HEK293A cells were
transfected to coexpress HA-melanophilin and either FLAG-syntaxin-4 or its truncated mutants
shown in the diagram. The cell lysates were incubated with anti-FLAG beads, and the bound
proteins and 1/60 volume of the reaction mixture were analyzed by immunoblotting with anti-
HA or anti-FLAG antibodies. (D) HEK293A cells were transfected to coexpress HA-syntaxin-4
and either FLAG-melanophilin or its truncated mutants as shown in the diagram. The FLAG-
immunoprecipitates were analyzed as in (C). (E) HEK293A cells were transfected to express
27 Diabetes Page 28 of 55
syntaxin-4 and/or the OSF-tagged, N-terminal melanophilin (1-400 residues). The N-terminal melanophilin and the binding proteins were pulled down using Strept-Tactin beads, and were subjected to SDS-PAGE and Coomassie Brilliant Blue staining. (F) MIN6 cells were transfected to express HA-melanophilin (1-146 residues) and FLAG-syntaxin-4 (1-273 residues), and their interaction was examined as in (C). (G) Glutathione S-transferase (GST)-fused melanophilin (1-
146 residues) and maltose binding protein (MBP)-fused syntaxin-4 (1-273 residues) were expressed in E. coli and were affinity-purified. Because intact melanophilin proteins were hardly expressed in bacteria (see Supplementary Fig. 8), coimmunoprecipitation experiments were performed between syntaxin-4 (0.5 μg) and melanophilin with an excess of degradation products
(total 20 μg). (H, I) HEK293A cells were transfected to coexpress FLAG-syntaxin-4 and mCherry-fused, N-terminal fragments of melanophilin (H), or to coexpress FLAG-tagged wild- type or Y122A/H124A/K130A melanophilin with HA-syntaxin-4 and HA-Rab27a (I). The
FLAG-immunoprecipitates were analyzed by immunoblotting with anti-red fluorescent protein, anti-HA, or anti-FLAG antibodies.
Figure 5. Melanophilin mutant defective in binding with Rab27a, syntaxin-4, or myosin-Va fails to restore the decreased insulin secretion in leaden β cells
(A) MIN6 cells were infected with adenovirus encoding FLAG-tagged, wild-type melanophilin, or its mutant: Y122A/H124A/K130A, E14A, or D378A/E380A/E381A/E382A/A467P. The immunoprecipitates with anti-FLAG antibody, as well as 1/100 volume of the original lysates, were electrophoresed and immunoblotted with the indicated antibodies. (B) Leaden pancreatic β cells were infected with adenoviruses encoding FLAG- or HA-tagged, wild-type or mutant melanophilin. They were coimmunostained with rabbit FLAG and mouse anti-Rab27a antibodies
28 Page 29 of 55 Diabetes
(middle), or with rat HA antibody and either rabbit anti-syntaxin-4 (upper) or anti-myosin-Va
antibodies (lower). Insets represent higher magnification micrographs of cells within the region
outlined by frames. Bars, 5 μm. (C) The wild-type and leaden islets were infected with
adenoviruses encoding LacZ or FLAG-melanophilin. After a 1 h infection, the islets were rinsed
and incubated for 48 h at 37ºC. The protein level of wild-type FLAG-melanophilin in leaden
islets were adjusted to that of endogenous melanophilin in wild-type islets by immunoblotting
with anti-melanophilin antibody (upper left). The protein levels of syntaxin-4 normalized by
those of β-actin were measured by densitometry (upper right: n = 3; P = 0.052, Student’s t-test).
Then the protein levels of mutant FLAG-melanophilin expressed in leaden islets were matched
to that of wild-type FLAG-melanophilin expressed at the endogenous level by immunoblotting
with anti-FLAG antibody (lower). (D) The wild-type and leaden islets were infected with
adenoviruses with the condition described in (C). The islets were preincubated in 2.8 mM
glucose-containing KRB buffer for 1 h, and were incubated in either 2.8 (LG, open dots) or 25
mM (HG, closed dots) glucose buffer for 1 h. Insulin levels secreted in the media and left in the
cell lysates were measured, and their ratios are shown (n = 6 from 6 mice). (E) TIRF microscopic
analysis of insulin granule exocytosis was performed as in Fig. 2 in leaden β cells expressing
Insulin-EGFP and either LacZ (white), FLAG- tagged, wild-type melanophilin (black), or its
mutant: Y122A/H124A/K130A (gray) or D378A/E380A/E381A/E382A/A467P (purple; n = 15
cells from 5 mice each). #P < 0.05, ##P < 0.01, ###P < 0.001; one-way ANOVA.
Figure 6. Melanophilin interacts with the open form of syntaxin-4 and myosin-Va in Ca2+-
dependent manners in cells
(A) HEK293A cells were transfected to express FLAG-melanophilin and either HA-syntaxin-4
29 Diabetes Page 30 of 55
wild type or the L173A/E174A mutant that adopts a constitutively open conformation. The cell lysates were subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with anti-HA and anti-FLAG antibodies, and were analyzed as in Fig. 4D. (B-F)
MIN6 cells (B, C), those expressing LacZ, FLAG-syntaxin-4 wild type or the L173A/E174A mutant (D), or those expressing HA-melanophilin (E,F) were incubated in non-stimulatory KRB buffer containing 2.8 mM glucose and 2 mM CaCl2 for 1 h at 37ºC. They were then stimulated with 25 mM glucose for 0, 3, or 30 min (B, E), for 30 min (C,D), or for 3 min (F), in the absence or presence of 10 mM EGTA as indicated. The cells were lysed, and the lysates were subjected to immunoprecipitation with control IgG (C), anti-syntaxin-4 (B, C), anti-FLAG (D), or anti-HA
(E,F) antibody. The immunoprecipitate, as well as 1:100 of the original lysates, were immunoblotted with the indicated antibodies. We expressed HA-melanophilin (E,F), because the anti-melanophilin antibody was unable to precipitate endogenous melanophilin, and because the anti-myosin-Va antibody was unable to coprecipitate endogenous melanophilin possibly due to its epitope interference. Note that the association of melanophilin with syntaxin-4 continuously increased during 30 min of glucose stimulation (B), whereas that with myosin-Va and actin transiently decreased at 3 min but was recovered at 30 min after glucose stimulation (E). P <
0.05; Student’s t-test.
30 Page 31 of 55 Diabetes
A 200 110 25 ** * 175 100 20 150 90 15 * 80 125 10 70 100 5 60 Body weight (g) Glucose (mg/dl) 75 50 0 40 50 % of initial glucose level WT 0 30 60 90 120 0 15 30 45 60 Time (min) Leaden Time (min) B Pituitary Liver Muscle Fat Islet
WT WT WT WT WT Leaden Leaden Leaden Leaden Leaden kDa Melano -philin 75
GAPDH 37
C b cells Leaden WT Leaden HA- LacZ LacZ Melanophilin kDa
Melanophilin 75 50 b-actin
D HA- Melanophilin Insulin Merge
E 16.7 mM Glucose 10 mM Forskolin 0.3 0.7 * *** 0.6
0.5 0.2 0.4
0.3 0.1 0.2 Insulin secretion Insulin secretion (% of insulin content) (% of insulin content) 0.1
0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 Time (min) Time (min)
Figure 1 B A D C
Number of fusion events Number of fusion events Number of fusion events (/200 mm2/20 min) 0.5 1.5 2.5 (/200mm2/20min) 0.5 1.5 2.5
10 12 14 16 (/200 mm2/20 min) 0 2 4 6 8 0 1 2 3 0 1 2 3 0-1 0-1 Resident 1-2 1-2
2-3 2-3 First phase First phase
3-4 3-4 First phase Visitor (1-7 min) 4-5 4-5
5-6 5-6
6-7 6-7 Passenger
7-8 7-8
8-9 8-9 Time (min)
Time (min) 9-10 9-10 10-11 10-11 Relative intensity Relative intensity Total 0 1 2 3 4 0 1 2 3 4 11-12 11-12 Passengers Residents Time (sec) -0.1 Time (sec) -0.1 12-13 12-13 Second phase 0 0 Second phase 0.1 0.1 13-14 13-14 0.2 0.2 14-15 0.3 0.3 14-15 0.4 0.4 Number of fusion events 15-16 15-16
(/200mm2/20min Diabetes 10 12 14 16 E 16-17
0 2 4 6 8 16-17
Number of fusion events 17-18 Leaden 17-18 Resident (/200 mm2/20 min) 18-19 10 20 30 40 50 60 18-19 WT 0 shRNA/mCherry 19-20 19-20 Second phase R… (8-20 min) Visitor Passenger
V… Insulin-EGFP * Total
P… * * Merge T… * Page 32of55 Figure 2 Page 33 of 55 Diabetes
A 3.5 3 2.5 /min) 2 2 m m 1.5 1 (/800 0.5
Number of fusion events 0 0 100 200 300 400 500 600 700 800 900 1000 Time (s) B C ** *** ) 2 m m (/800 Sequential exocytosis Number of fusion events (% of total fusion events)
0-300s 300-600s 600-900s WT Leaden Figure 3 Diabetes Page 34 of 55
A IP: HA- B D (mutant construct) Input Melanophilin Input IP
GSLEWYYQHV
Antibodies 1 147 400 590 RBD MBD ABD HA-
HA- Melanophilin: LacZ LacZ 116-125 Syntaxin-4 Control IgG Control IgG Syntaxin-4 kDa Melanophilin Melanophilin kDa N terminal: 1 400 Melanophilin Melanophilin 75 75 C terminal: 147 590 Rab27a 25 37 116-400: 116 400 250 Syntaxin-4 Myosin-Va 126-400: 126 400 50 b-actin 147-400: 147 400 C (mutant construct) α-tubulin 50 FLAG-Melanophilin EB1 37 37 70 78 112 118163 194 273 298 Syntaxin-1a 37 Syntaxin-4: 1 Ha Hb Hc H3 TM 298
N-Habc: 1 (-) Syntaxin-2 37 193 (1-400) 126-400 147-400 116-400 (147-590) N terminal H3: 194 273 C terminal 37 kDa kDa Syntaxin-3 FLAG-Syntaxin-4 Syntaxin-4 37 37 Syntaxin-4 37 Input (-) WT H3
75 Habc kDa Syntaxin-4 37 37 Munc18-1 Melanophilin 50 75 75 Munc18-2 Input
75 IP: FLAG- Melanophilin Munc18-3 Melanophilin Melanophilin 75 50 SNAP25 25
VAMP2 15
25 E OSF-Melanophilin Syntaxin-4 H mCherry-Melanophilin
Melanophilin 20 - + + (-) 1-130 1-146 1-120 1-100 1-110 kDa N terminal (1-400) IP: FLAG-Syntaxin-4 Syntaxin-4 + + - Melanophilin
kDa Input 37 250 F Melanophilin 150 Input IP: FLAG 37 FLAG-Syntaxin-4 100 Syntaxin4
- + - + IP: FLAG- (1-273) Syntaxin-4 37 75 HA-Melanophilin + + + + Melanophilin (1-146) kDa HA-Melanophilin 50 I FLAG-Melanophilin (1-146) 20 FLAG-Syntaxin-4 (-) 37 37 WT
(1-273) K130A Y122A/ Syntaxin4 H124A/ kDa
G Input IP: MBP Syntaxin-4 37 Input 25 Rab27a 25 MBP MBP- MBP MBP- (1-273) (1-273) Syntaxin-4 Syntaxin-4 kDa GST-Melanophilin Syntaxin-4 37 (1-146) 37 Rab27a 25 MBP-Syntaxin-4
75 IP: FLAG- (1-273) Melanophilin Melanophilin 75
50 MBP Figure 4 Page 35 of 55 Diabetes
A FLAG-Melanophilin B C Islets Syntaxin-4 expression HA- WT Leaden
WT Leaden islets Melanophilin K130A Y122A/ H124A/ kDa WT Syntaxin-4 Merge p=0.052 Syntaxin-4 37 LacZ LacZ FLAG-
Melanophilin kDa Rab27a 25 Input Melanophilin 250 Myosin-Va 75 Y122A/ 37 H124A/ 37 Syntaxin-4 Syntaxin-4 K130A Syntaxin-4 Merge Relative to WT (LacZ) 50 Rab27a 25 b-actin LacZ FLAG- Melanophilin Myosin-Va 250 IP: FLAG- Leaden islets Melanophilin D378A/ Melanophilin 75 E380A/ Y122A/ E381A/ FLAG-Melanophilin H124A/ E382A/ LacZ WT E14A K130A A467P kDa FLAG-
WT FLAG- E14A Melanophilin Melanophilin 75 Syntaxin-4 50 WT Rab27a Merge b-actin
Rab27a Input D Islets Myosin-Va WT Leaden
Syntaxin-4 E14A Rab27a Merge
### # Rab27a # Myosin-Va IP: FLAG-
Melanophilin ## Melanophilin Insulin secretion (% of total insulin) FLAG-Melanophilin
LacZ LacZ WT E14A Y122A/ D378A/ HA- H124A/ E380A/ E381A/ WT Melanophilin K130A E382A/ A467P WT Myosin-Va Merge A467P E381A/E382A/ D378A/E380A/ FLAG-Melanophilin Syntaxin-4
E 30 Leaden b cells Rab27a ## # Input LacZ # D378A/ WT Myosin-Va E380A/ Y122A/H124A/K130A E381A/ 20 D378A/E380A/E381A/E382A/A467P E382A/ Syntaxin-4 A467P Myosin-Va Merge /20 min) 2 ### # m m # Rab27a 10 (/200
IP: FLAG- Myosin-Va Melanophilin Number of fusion events
Melanophilin 0 Residents Visitors Passengers Total Figure 5 Diabetes Page 36 of 55
HA-Syntaxin-4 A B IP: Syntaxin-4 WT L173A/E174A IP: Syntaxin-4
Glucose (25 mM) 0min 3 30 kDa Melanophilin Sintaxin-4 Melanophilin
(-) 75 (-) * * FLAG- FLAG- Syntaxin-4 37 Melanophilin Melanophilin kDa Input Syntaxin-4 37 Input Glucose (25 mM) 0min 3 30 kDa
Syntaxin-4 37 Melanophilin Relative to 0 min 75 Glucose 37 3 30 3 30 Melanophilin Syntaxin-4 (min) IP: FLAG-
Melanophilin 75 C IP: IP: D Input IP: FLAG-syntaxin-4 Input IgG Syntaxin-4 Glucose 2.8 25 2.8 (mM) 25 Glucose (25mM) -- + + -- + + EGTA (10mM) --- + --- + kDa Melanophilin 75 WT WT WT WT LacZ LacZ LacZ LacZ SNAP25 25 L173A/E174A L173A/E174A L173A/E174A L173A/E174A FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 FLAG-syntaxin-4 kDa VAMP2 15 Melanophilin 75 37 Syntaxin-4 Syntaxin-4 37 IP: Syntaxin-4 * Melanophilin SNAP25 VAMP2 Sintaxin-4 * * * * at Glucose 2.8 mM) (Relative to Syntaxin-4 WT Expression of Melanophilin Relative to Control L173A/ L173A/ without 25 mM Glucose WT WT FLAG-Syntaxin-4 E174A E174A Glucose (25 mM) + + + + + + + + Glucose (mM) 2.8 25 EDTA - + - + - + - + Input IP: HA-melanophilin E IP: F LacZ HA-melanophilin LacZ HA-melanophilin Input HA-Melanophilin Glucose (25mM) -- -- + + Glucose (25mM) 0min 3 30 0min 3 30 kDa + + EGTA (10mM) --- + -- - + kDa Myosin-Va 250 Myosin-Va 250 50 b-actin b-actin 50
Melanophilin 75 Melanophilin 75 IP: HA-Melanophilin IP: HA-Melanophilin
Myosin-Va b-actin Melanophilin Myosin-Va b-actin Melanophilin * * * * Relative to Control Relative to 0 min without 25 mM Glucose Glucose 3 30 3 30 3 30 Glucose (min) (25 mM) + + + + + + EDTA - + - + - + Figure 6 Page 37 of 55 Diabetes
!""#$%$&'(# )('(
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iabetes Page 38 of 55 &'+,(!2 P'$$ X%*!($%!* >(11%7 H20"%!D^( ['?3!0$0*2G QL ! Page 39 of 55 Diabetes !""#$%$&'(# ,(-#$ B/ >*5%$* 42* 3#2&5&<; %!'(<$&$151; (&6 C!(&'5'('5D$ >@A ,(*<$' >*5%$* $C!$&3$ H'$(!0)3%$%! A#$$ $'!*73 =0+`(+@ &&&&AA[[PA[&&&&AAAA&&[[&&APP[ >'6'+"' &&&P[P&A&[[A&&&P[&P[&&&PPA[P H'$(!0)3%$%! A+(*,'!7 E 49DL<<8 =0+`(+@ &&&&AA[[PA[&&&&AAAA&&[[&&APP[ >'6'+"' &&&P[P&A&[[A&&[&P[&&AAPPAP[&A[& H'$(!0)3%$%! A+(*,'!7 99UDL<< =0+`(+@ &&&&AA[[PA[&&&[[P[P[&&A&[&&[AP[A H'$(!0)3%$%! A+(*,'!7 9NUDL<< =0+`(+@ &&&&AA[[PA[&A&&&P[P&P[[PAA&P&[[ H'$(!0)3%$%! A+(*,'!7 P 49LOD;B<8 =0+`(+@ &&&&AA[[PA[&&&[&&A&&[&&A[P[&A&P >'6'+"' &&&P[P&A&[[A&&&P[&P[&&&PPA[P H'$(!0)3%$%! A+(*,'!7 9D9<< =0+`(+@ &&&P[P&A&P[A[&&&&AAAA&&[[&&APP[ >'6'+"' &&&&AA[[P[[AP[&P[P[[P[&&&[&&&P H'$(!0)3%$%! A+(*,'!7 9D99< >'6'+"' &&&&AA[[P[[A&&PPA&&[&&PA&&&&[P H'$(!0)3%$%! A+(*,'!7 9D9N< >'6'+"' &&&&AA[[P[[APPAP[PPA&A&AAPP&A[ H'$(!0)3%$%! A+(*,'!7 9D9Q< >'6'+"' &&&&AA[[P[[AP[[&AA&P&A&PPP[PAP H'$(!0)3%$%! A+(*,'!7 9D9LU >'6'+"' &&&&AA[[P[[AP[&PA&PP&PPPAPA&A& H'$(!0)3%$%! ,#7(7%0! I9LA =0+`(+@ APA&AP&A&&P&&P[&A&PAP >'6'+"' &[&P[PA&PP&PP[P&[P[&[ H'$(!0)3%$%! ,#7(7%0! =0+`(+@ PPPA&[&P[&PPAPA&P&&P&&P&APAP[PA&& VQORA:IQR H'$(!0)3%$%! ,#7(7%0! ALUOW =0+`(+@ &[[PA&PA&PP[&A&A&[&A& >'6'+"' P[PAP[P[PA&&P[&P[&AAP H'$(!0)3%$%! ,#7(7%0! a9NNA =0+`(+@ &A&[&&[AP&PPPA&PAP&[&A&& >'6'+"' PP[PAP&[&P[&&&P&[APPAP[P