Mutations in MYLPF Cause a Novel Segmental Amyoplasia that Manifests as Distal Arthrogryposis.

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

Nanosurfer assay dissects ß-cardiac myosin and cardiac myosin-binding protein C interactions.

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a "nanosurfer" assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human ß-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0-C2 or C1-C2) on nanotubes every 14 nm. Both C0-C2 and C1-C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0-C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0-C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of ß-cardiac myosin contractility.

Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding.

Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

Skeletal MyBP-C isoforms tune the molecular contractility of divergent skeletal muscle systems.

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein, localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle type-specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, 4 MyBP-C isoforms (1 fast-type MyBP-C and 3 N-terminally spliced slow-type MyBP-C) were expressed in EDL, but only the 3 slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that, only in the C-zone, MyBP-C Ca2+ sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C's N terminus as N-terminal proteolysis attenuated MyBP-C's functional capacities. To determine each MyBP-C isoform's contribution to thin filament Ca2+ sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoform's N terminus is functionally distinct and has modulatory capacities that depend on the muscle type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.

Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time.

Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, "relaxed" muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing "relaxed" population, and a slower-hydrolyzing "super relaxed" (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.

Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism.

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Myosin VI has a one track mind versus myosin Va when moving on actin bundles or at an intersection.

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin-actin) or mixed polarity (α-actinin-actin) bundles. The stepping dynamics of individual Qdot-labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development, and promoting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility from the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting MyBP-H may be functionally silent. However, our results suggest an active role. Small angle x-ray diffraction of intact larval tails revealed MyBP-H contributes to the compression of the myofilament lattice accompanying stretch or contraction, while in vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake". These results provide new insights and raise questions about the role of the C-zone during muscle development.

"Spatial Relationships Matter: Kinesin-1 Molecular Motors Transport Liposome Cargo Through 3D Microtubule Intersections In Vitro".

Kinesin-1 ensembles maneuver vesicular cargoes through intersections in the 3-dimensional (3D) intracellular microtubule (MT) network. To characterize directional outcomes (straight, turn, terminate) at MT intersections, we challenge 350 nm fluid-like liposomes transported by ~10 constitutively active, truncated kinesin-1 KIF5B (K543) with perpendicular 2-dimensional (2D) and 3D intersections in vitro. Liposomes frequently pause at 2D and 3D intersections (~2s), suggesting that motor teams can simultaneously engage each MT and undergo a tug-of-war. Once resolved, the directional outcomes at 2D MT intersections have a straight to turn ratio of 1.1; whereas at 3D MT intersections, liposomes more frequently go straight (straight to turn ratio of 1.8), highlighting that spatial relationships at intersections bias directional outcomes. Using 3D super-resolution microscopy (STORM), we define the gap between intersecting MTs and the liposome azimuthal approach angle heading into the intersection. We develop an in silico model in which kinesin-1 motors diffuse on the liposome surface, simultaneously engage the intersecting MTs, generate forces and detach from MTs governed by the motors' mechanochemical cycle, and undergo a tug-of-war with the winning team determining the directional outcome in 3D. The model predicts that 1-3 motors typically engage the MT, consistent with optical trapping measurements. Modeled liposomes also predominantly go straight through 3D intersections over a range of intersection gaps and liposome approach angles, even when obstructed by the crossing MT. Our observations and modeling offer mechanistic insights into how cells might tune the MT cytoskeleton, cargo, and motors to modulate cargo transport.

Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle.

The myosin super-relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with myosin-binding protein C (MyBP-C) in the sarcomere C-zone. To define how cardiac MyBP-C (cMyBP-C) and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic cMyBP-C null mice and those expressing cMyBP-C with a 271-residue N-terminal truncation. Utilizing super-resolution microscopy, we determined the lifetime and subsarcomeric location of individual fluorescent-ATP turnover events within isolated cardiac myofibrils. The proportion of SRX myosin demonstrated a gradient along the half-thick filament, highest in the P- and C-zones (72 ± 9% and 71 ± 6%, respectively) and lower in the D-zone (45 ± 10%), which lies farther from the sarcomere center and lacks cMyBP-C, suggesting a possible role for cMyBP-C in stabilizing the SRX. However, myofibrils from cMyBP-C null mice demonstrated an ∼40% SRX reduction, not only within the now cMyBP-C-free C-zone (49 ± 9% SRX), but also within the D-zone (22 ± 5% SRX). These data suggest that the influence of cMyBP-C on the SRX state is not limited to the C-zone but extends along the thick filament. Interestingly, myofibrils with N-terminal truncated cMyBP-C had an SRX content and spatial gradient similar to the cMyBP-C null, indicating that the N terminus of cMyBP-C is necessary for cMyBP-C's role in enhancing the SRX gradient along the entire thick filament. Given that SRX myosin exist as a gradient along the thick filament that is highest in the C-zone, even in the absence of cMyBP-C or its N-terminus, an inherent bias must exist in the structure of the thick filament to stabilize the SRX state.

The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner.

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0-C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1-C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0-C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0-C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0-C3 in proportion to the total number phosphomimetic replacements. Under load, C0-C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0-C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0-C3 on F:V was well fitted by a model that assumed C0-C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.

Diabetes with heart failure increases methylglyoxal modifications in the sarcomere, which inhibit function.

Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.

Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner.

Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.

Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling.

The beating heart exhibits remarkable contractile fidelity over a lifetime, which reflects the tight coupling of electrical, chemical, and mechanical elements within the sarcomere, the elementary contractile unit. On a beat-to-beat basis, calcium is released from the ends of the sarcomere and must diffuse toward the sarcomere center to fully activate the myosin- and actin-based contractile proteins. The resultant spatial and temporal gradient in free calcium across the sarcomere should lead to nonuniform and inefficient activation of contraction. We show that myosin-binding protein C (MyBP-C), through its positioning on the myosin thick filaments, corrects this nonuniformity in calcium activation by exquisitely sensitizing the contractile apparatus to calcium in a manner that precisely counterbalances the calcium gradient. Thus, the presence and correct localization of MyBP-C within the sarcomere is critically important for normal cardiac function, and any disturbance of MyBP-C localization or function will contribute to the consequent cardiac pathologies.

Cytoskeletal dependence of insulin granule movement dynamics in INS-1 beta-cells in response to glucose.

For pancreatic ß-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 ß-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.