On the Challenges of Identifying Benthic Dominance on Anthropocene Coral Reefs.

The concept of dominance is frequently used to describe changes in rapidly reconfiguring ecosystems, but the definition of dominance can vary widely among studies. Using coral reefs as a model, we use extensive benthic composition data to explore how variability in applying dominance concepts can shape perceptions. We reveal that coral dominance is sensitive to the exclusion of key algal groups and the categorization of other benthic groups, with ramifications for detecting an ecosystem phase shift. For example, ignoring algal turf inflates the dominance of hard and soft corals in the benthic habitats underpinning reef ecosystems. We need a consensus on how dominance concepts are applied so that we can build a more comprehensive understanding of ecosystem shifts across a broad range of aquatic and terrestrial settings. For reefs, we highlight the benefits of comprehensive and inclusive surveys for evaluating and managing the altered ecosystem states that are emerging in the Anthropocene.

Nucleotide exchange factor Rab3GEP requires DENN and non-DENN elements for activation and targeting of Rab27a.

Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

Long-term dynamics and drivers of coral and macroalgal cover on inshore reefs of the Great Barrier Reef Marine Park.

Quantifying the role of biophysical and anthropogenic drivers of coral reef ecosystem processes can inform management strategies that aim to maintain or restore ecosystem structure and productivity. However, few studies have examined the combined effects of multiple drivers, partitioned their impacts, or established threshold values that may trigger shifts in benthic cover. Inshore fringing reefs of the Great Barrier Reef Marine Park (GBRMP) occur in high-sediment, high-nutrient environments and are under increasing pressure from multiple acute and chronic stressors. Despite world-leading management, including networks of no-take marine reserves, relative declines in hard coral cover of 40-50% have occurred in recent years, with localized but persistent shifts from coral to macroalgal dominance on some reefs. Here we use boosted regression tree analyses to test the relative importance of multiple biophysical drivers on coral and macroalgal cover using a long-term (12-18 yr) data set collected from reefs at four island groups. Coral and macroalgal cover were negatively correlated at all island groups, and particularly when macroalgal cover was above 20%. Although reefs at each island group had different disturbance-and-recovery histories, degree heating weeks (DHW) and routine wave exposure consistently emerged as common drivers of coral and macroalgal cover. In addition, different combinations of sea-surface temperature, nutrient and turbidity parameters, exposure to high turbidity (primary) floodwater, depth, grazing fish density, farming damselfish density, and management zoning variously contributed to changes in coral and macroalgal cover at each island group. Clear threshold values were apparent for multiple drivers including wave exposure, depth, and degree heating weeks for coral cover, and depth, degree heating weeks, chlorophyll a, and cyclone exposure for macroalgal cover, however, all threshold values were variable among island groups. Our findings demonstrate that inshore coral reef communities are typically structured by broadscale climatic perturbations, superimposed upon unique sets of local-scale drivers. Although rapidly escalating climate change impacts are the largest threat to coral reefs of the GBRMP and globally, our findings suggest that proactive management actions that effectively reduce chronic stressors at local scales should contribute to improved reef resistance and recovery potential following acute climatic disturbances.

Early recovery dynamics of turbid coral reefs after recurring bleaching events.

The worlds' coral reefs are declining due to the combined effects of natural disturbances and anthropogenic pressures including thermal coral bleaching associated with global climate change. Nearshore corals are receiving increased anthropogenic stress from coastal development and nutrient run-off. Considering forecast increases in global temperatures, greater understanding of drivers of recovery on nearshore coral reefs following widespread bleaching events is required to inform management of local stressors. The west Pilbara coral reefs, with cross-shelf turbidity gradients coupled with a large nearby dredging program and recent history of repeated coral bleaching due to heat stress, represent an opportune location to study recovery from multiple disturbances. Mean coral cover at west Pilbara reefs was monitored from 2009 to 2018 and declined from 45% in 2009 to 5% in 2014 following three heat waves. Recruitment and juvenile abundance of corals were monitored from 2014 to 2018 and were combined with biological and physical data to identify which variables enhanced or hindered early-stage coral recovery of all hard corals and separately for the acroporids, the genera principally responsible for recovery in the short-term (<7 years). From 2014 to 2018, coral cover increased from 5 to 10% but recovery varied widely among sites (0-13%). Hard coral cover typically recovered most at shallower sites that had higher abundance of herbivorous fish, less macroalgae, and lower turbidity. Similarly, acroporid corals recovered most at sites with lower turbidity and macroalgal cover. Juvenile acroporid densities were a good indicator of recovery at least two years after they were recorded. However, recruitment to settlement tiles was not a good predictor of total coral or acroporid recovery. This study shows that coral recovery can be slower in areas of high turbidity and the rate may be reduced by local pressures, such as dredging. Management should focus on improving or maintaining local water quality to increase the likelihood of coral recovery under climate stress. Further, in turbid environments, juvenile coral density predicts early coral recovery better than recruits on tiles and may be a more cost-effective technique for monitoring recovery potential.

Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport.

Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here, we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes.

Seascape genomics reveals fine-scale patterns of dispersal for a reef fish along the ecologically divergent coast of Northwestern Australia.

Understanding the drivers of dispersal among populations is a central topic in marine ecology and fundamental for spatially explicit management of marine resources. The extensive coast of Northwestern Australia provides an emerging frontier for implementing new genomic tools to comparatively identify patterns of dispersal across diverse and extreme environmental conditions. Here, we focused on the stripey snapper (Lutjanus carponotatus), which is important to recreational, charter-based and customary fishers throughout the Indo-West Pacific. We collected 1,016 L. carponotatus samples at 51 locations in the coastal waters of Northwestern Australia ranging from the Northern Territory to Shark Bay and adopted a genotype-by-sequencing approach to test whether realized connectivity (via larval dispersal) was related to extreme gradients in coastal hydrodynamics. Hydrodynamic simulations using CONNIE and a more detailed treatment in the Kimberley Bioregion provided null models for comparison. Based on 4,402 polymorphic single nucleotide polymorphism loci shared across all individuals, we demonstrated significant genetic subdivision between the Shark Bay Bioregion in the south and all locations within the remaining, more northern bioregions. More importantly, we identified a zone of admixture spanning a distance of 180 km at the border of the Kimberley and Canning bioregions, including the Buccaneer Archipelago and adjacent waters, which collectively experiences the largest tropical tidal range and some of the fastest tidal currents in the world. Further testing of the generality of this admixture zone in other shallow water species across broader geographic ranges will be critical for our understanding of the population dynamics and genetic structure of marine taxa in our tropical oceans.

Restricted gene flow and local adaptation highlight the vulnerability of high-latitude reefs to rapid environmental change.

Global climate change poses a serious threat to the future health of coral reef ecosystems. This calls for management strategies that are focused on maximizing the evolutionary potential of coral reefs. Fundamental to this is an accurate understanding of the spatial genetic structure in dominant reef-building coral species. In this study, we apply a genotyping-by-sequencing approach to investigate genome-wide patterns of genetic diversity, gene flow, and local adaptation in a reef-building coral, Pocillopora damicornis, across 10 degrees of latitude and a transition from temperate to tropical waters. We identified strong patterns of differentiation and reduced genetic diversity in high-latitude populations. In addition, genome-wide scans for selection identified a number of outlier loci putatively under directional selection with homology to proteins previously known to be involved in heat tolerance in corals and associated with processes such as photoprotection, protein degradation, and immunity. This study provides genomic evidence for both restricted gene flow and local adaptation in a widely distributed coral species, and highlights the potential vulnerability of leading-edge populations to rapid environmental change as they are locally adapted, reproductively isolated, and have reduced levels of genetic diversity.

Novel hypoglycemic injury mechanism: N-methyl-D-aspartate receptor-mediated white matter damage.

OBJECTIVE: Hypoglycemia is a common adverse event and can injure central nervous system (CNS) white matter (WM). We determined whether glutamate receptors were involved in hypoglycemic WM injury. METHODS: Mouse optic nerves (MON), CNS WM tracts, were maintained at 37°C with oxygenated artificial cerebrospinal fluid (ACSF) containing 10mM glucose. Aglycemia was produced by switching to 0 glucose ACSF. Supramaximal compound action potentials (CAPs) were elicited using suction electrodes, and axon function was quantified as the area under the CAP. Amino acid release was measured using high-performance liquid chromatography. Extracellular lactate concentration ([lactate(-)]o) was measured using an enzyme electrode. RESULTS: About 50% of MON axons were injured after 60 minutes of aglycemia (90% after 90 minutes); injury extent was not affected by animal age. Blockade of N-methyl-D-aspartate (NMDA)-type glutamate receptors improved recovery after 90 minutes of aglycemia by 250%. Aglycemic injury was increased by reducing [Mg(2+)]o or increasing [glycine]o , and decreased by lowering pHo , expected results for NMDA receptor-mediated injury. pHo increased during aglycemia due to a drop in [lactate(-)]o. Aglycemic injury was dramatically reduced in the absence of [Ca(2+)]o. Extracellular aspartate, a selective NMDA receptor agonist, increased during aglycemia ([glutamate]o fell). INTERPRETATION: Aglycemia injured WM by a unique excitotoxic mechanism involving NMDA receptors (located primarily on oligodendrocytes). During WM aglycemia, the selective NMDA agonist aspartate is released, probably from astrocytes. Injury is mediated by Ca(2+) influx through aspartate-activated NMDA receptors made permeable by an accompanying alkaline shift in pHo caused by a fall in [lactate(-)]o. These insights have important clinical implications.

Can juvenile supply predict future abundance of large-bodied reef fishes?

The extent to which juvenile abundance can predict future populations of lethrinids at Ningaloo Reef was assessed using size frequency data collected over 13 consecutive years. Annual abundance of juvenile lethrinids (<5 cm TL) was highest in northern Ningaloo during La Niña years, when seawater is warmer and oceanic currents stronger. Juvenile lethrinid abundance explained 35% of the variance in 1-2 year-old Lethrinus nebulosus abundance the following year, a steeper relationship in the north suggesting greater survival of juveniles. Juvenile lethrinid abundance was also positively correlated to abundance of 1-2 year-old L. atkinsoni in the southern region of Ningaloo. Abundance of juvenile lethrinids were however poor predictors of L. nebulosus and L. atkinsoni older than 2 years of age. Post settlement processes likely weaken the link between juvenile supply and abundance of lethrinids >2 years old making it difficult to accurately quantify the overall size of future lethrinid populations.

Glycogen function in adult central and peripheral nerves.

We studied the roles of glycogen in axonal pathways of the central nervous system (CNS) and peripheral nervous system (PNS). By using electrophysiological recordings, in combination with biochemical glycogen assay, it was possible to determine whether glycogen was crucial to axon function under different conditions. Glycogen was present both in mouse optic nerve (MON) and in mouse sciatic nerve (MSN). Aglycemia caused loss of the compound action potential (CAP) in both pathways after a latency of 15 min (MON) and 120 min for myelinated axons (A fibers) in the MSN. With the exception of unmyelinated axons (C fibers) in the MSN, CAP decline began when usable glycogen was exhausted. Glycogen was located in astrocytes in the MON and in myelinating Schwann cells in the MSN; it was absent from the Schwann cells surrounding unmyelinated C fibers. In MON, astrocytic glycogen is metabolized to lactate and "shuttled" to axons to support metabolism. The ability of lactate to support A fiber conduction in the absence of glucose suggests a common pathway in both the CNS and the PNS. Lactate is released from MON and MSN in substantial quantities. That lactate levels fall in MSN in the presence of diaminobenzidine, which inhibits glycogen phosphorylase, strongly suggests that glycogen metabolism contributes to lactate release under resting conditions. Glycogen is a "backup" energy substrate in both the CNS and the PNS and, beyond sustaining excitability during glucose deprivation, has the capacity to subsidize the axonal energy demands during times of intense activity in the presence of glucose.

Schwann cell glycogen selectively supports myelinated axon function.

OBJECTIVE: Interruption of energy supply to peripheral axons is a cause of axon loss. We determined whether glycogen was present in mammalian peripheral nerve, and whether it supported axon conduction during aglycemia. METHODS: We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. RESULTS: Glycogen was present in sciatic nerve, its concentration varying directly with ambient glucose. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm, and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time course of glycogen loss. Latency to compound action potential (CAP) failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small-diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. INTERPRETATION: Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large-diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. .

Adaptive management of the Great Barrier Reef: a globally significant demonstration of the benefits of networks of marine reserves.

The Great Barrier Reef (GBR) provides a globally significant demonstration of the effectiveness of large-scale networks of marine reserves in contributing to integrated, adaptive management. Comprehensive review of available evidence shows major, rapid benefits of no-take areas for targeted fish and sharks, in both reef and nonreef habitats, with potential benefits for fisheries as well as biodiversity conservation. Large, mobile species like sharks benefit less than smaller, site-attached fish. Critically, reserves also appear to benefit overall ecosystem health and resilience: outbreaks of coral-eating, crown-of-thorns starfish appear less frequent on no-take reefs, which consequently have higher abundance of coral, the very foundation of reef ecosystems. Effective marine reserves require regular review of compliance: fish abundances in no-entry zones suggest that even no-take zones may be significantly depleted due to poaching. Spatial analyses comparing zoning with seabed biodiversity or dugong distributions illustrate significant benefits from application of best-practice conservation principles in data-poor situations. Increases in the marine reserve network in 2004 affected fishers, but preliminary economic analysis suggests considerable net benefits, in terms of protecting environmental and tourism values. Relative to the revenue generated by reef tourism, current expenditure on protection is minor. Recent implementation of an Outlook Report provides regular, formal review of environmental condition and management and links to policy responses, key aspects of adaptive management. Given the major threat posed by climate change, the expanded network of marine reserves provides a critical and cost-effective contribution to enhancing the resilience of the Great Barrier Reef.

Physical, biological and anthropogenic drivers of spatial patterns of coral reef fish assemblages at regional and local scales.

Species abundance, diversity and community assemblage structure are determined by multiple physical, habitat and management drivers that operate across multiple spatial scales. Here we used a multi-scale coral reef monitoring dataset to examine regional and local differences in the abundance, species richness and composition of fish assemblages in no-take marine reserve (NTMR) and fished zones at four island groups in the Great Barrier Reef Marine Park, Australia. We applied boosted regression trees to quantify the influence of 20 potential drivers on the coral reef fish assemblages. Reefs in two locations, Magnetic Island and the Keppel Islands, had distinctive fish assemblages and low species richness, while the Palm and Whitsunday Islands had similar species composition and higher species richness. Overall, our analyses identified several important physical (temperature, wave exposure) and biological (coral, turf, macroalgal and unconsolidated substratum cover) drivers of inshore reef fish communities, some of which are being altered by human activities. Of these, sea surface temperature (SST) was more influential at large scales, while wave exposure was important both within and between island groups. Species richness declined with increasing macroalgal cover and exposure to cyclones, and increased with SST. Species composition was most strongly influenced by mean SST and percent cover of macroalgae. There was substantial regional variation in the local drivers of spatial patterns. Although NTMR zoning influenced total fish density in some regions, it had negligible effects on fish species richness, composition and trophic structure because of the relatively small number of species targeted by the fishery. These findings show that inshore reef fishes are directly influenced by disturbances typical of the nearshore Great Barrier Reef, highlighting the need to complement global action on climate change with more targeted localised efforts to maintain or improve the condition of coral reef habitats.

Extinction vulnerability of coral reef fishes.

With rapidly increasing rates of contemporary extinction, predicting extinction vulnerability and identifying how multiple stressors drive non-random species loss have become key challenges in ecology. These assessments are crucial for avoiding the loss of key functional groups that sustain ecosystem processes and services. We developed a novel predictive framework of species extinction vulnerability and applied it to coral reef fishes. Although relatively few coral reef fishes are at risk of global extinction from climate disturbances, a negative convex relationship between fish species locally vulnerable to climate change vs. fisheries exploitation indicates that the entire community is vulnerable on the many reefs where both stressors co-occur. Fishes involved in maintaining key ecosystem functions are more at risk from fishing than climate disturbances. This finding is encouraging as local and regional commitment to fisheries management action can maintain reef ecosystem functions pending progress towards the more complex global problem of stabilizing the climate.

Rapid increase in fish numbers follows creation of world's largest marine reserve network.

No-take marine reserves (NTMRs) are much advocated as a solution to managing marine ecosystems, protecting exploited species and restoring natural states of biodiversity [1,2]. Increasingly, it is becoming clear that effective marine conservation and management at ecosystem and regional scales requires extensive networks of NTMRs [1,2]. The world's largest network of such reserves was established on Australia's Great Barrier Reef (GBR) in 2004. Closing such a large area to all fishing has been socially and politically controversial, making it imperative that the effectiveness of this new reserve network be assessed. Here we report evidence, first, that the densities of the major target species of the GBR reef line fisheries were significantly higher in the new NTMRs, compared with fished sites, in just two years; and second, that the positive differences were consistent for multiple marine reserves over an unprecedented spatial scale (>1,000 km).

Coral larval recruitment in north-western Australia predicted by regional and local conditions.

Understanding ecological processes that shape contemporary and future communities facilitates knowledge-based environmental management. In marine ecosystems, one of the most important processes is the supply of new recruits into a population. Here, we investigated spatiotemporal variability in coral recruitment at 15 reefs throughout the Dampier Archipelago, north-western Australia between 2015 and 2017 and identified the best environmental predictors for coral recruitment patterns over this period. Large differences in recruitment were observed among years with the average density of recruits increasing by 375% from 0.017 recruits cm-2 in 2015 to 0.059 recruits cm-2 in 2017. Despite differences in recruitment among years, the rank order of coral recruit density among reefs remained similar among years, suggesting that spatial variation in recruitment within the Dampier Archipelago is partly deterministic and predictable. The density of coral recruits was best explained by percent cover of live corals at both local (within 5 m) and meso-scales (within 15 km), water turbidity and an oceanographic model that predicted larval dispersal. The highest density of coral recruits (~0.13 recruits cm-2 or 37 recruits per tile) occurred on reefs within sub-regions (15 km) with greater than 35% coral cover, low to moderate turbidity (KD490 < 0.2) and moderate to high modelled predictions of larval dispersal. Our results demonstrate that broad-scale larval dispersal models, when combined with local metrics of percent hard coral cover and water turbidity, can reliably predict the relative abundance of coral recruits over large geographical areas and thus can identify hotspots of recruit abundance and potential recovery following environmental disturbances; information that is essential for effective management of coral reefs.

A tumor-associated beta 1 integrin mutation that abrogates epithelial differentiation control.

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type beta 1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the beta1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 beta 1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner alpha subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type beta1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of beta 1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.

Cell surface collagenolysis requires homodimerization of the membrane-bound collagenase MT1-MMP.

Pericellular degradation of interstitial collagens is a crucial event for cells to migrate through the dense connective tissue matrices, where collagens exist as insoluble fibers. A key proteinase that participates in this process is considered to be membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP-14), but little is known about the mechanism by which it cleaves the insoluble collagen. Here we report that homodimerization of MT1-MMP through its hemopexin (Hpx) domain is essential for cleaving type I collagen fibers at the cell surface. When dimerization was blocked by coexpressing either a membrane-bound or a soluble form of the Hpx domain, cell surface collagenolytic activity was inhibited in a dose-dependent manner. When MMP-13, a soluble collagenase active as a monomer in solution, was expressed as a membrane-anchored form on the cell surface, homodimerization was also required to cleave collagen. Our results introduce a new concept in that pericellular collagenolysis is regulated by correct molecular assembly of the membrane-anchored collagenase, thereby governing the directionality of the cell to migrate in tissue.

Hypothermic neuroprotection during reperfusion following exposure to aglycemia in central white matter is mediated by acidification.

Hypoglycemia is a common iatrogenic consequence of type 1 diabetes therapy that can lead to central nervous system injury and even death if untreated. In the absence of clinically effective neuroprotective drugs we sought to quantify the putative neuroprotective effects of imposing hypothermia during the reperfusion phase following aglycemic exposure to central white matter. Mouse optic nerves (MONs), central white matter tracts, were superfused with oxygenated artificial cerebrospinal fluid (aCSF) containing 10 mmol/L glucose at 37°C. The supramaximal compound action potential (CAP) was evoked and axon conduction was assessed as the CAP area. Extracellular lactate was measured using an enzyme biosensor. Exposure to aglycemia, simulated by omitting glucose from the aCSF, resulted in axon injury, quantified by electrophysiological recordings, electron microscopic analysis confirming axon damage, the extent of which was determined by the duration of aglycemia exposure. Hypothermia attenuated injury. Exposing MONs to hypothermia during reperfusion resulted in improved CAP recovery compared with control recovery measured at 37°C, an effect attenuated in alkaline aCSF. Hypothermia decreases pH implying that the hypothermic neuroprotection derives from interstitial acidification. These results have important clinical implications demonstrating that hypothermic intervention during reperfusion can improve recovery in central white matter following aglycemia.

The adaptor protein melanophilin regulates dynamic myosin-Va:cargo interaction and dendrite development in melanocytes.

The regulation of organelle transport by the cytoskeleton is fundamental for eukaryotic survival. Cytoskeleton motors are typically modular proteins with conserved motor and diverse cargo-binding domains. Motor:cargo interactions are often indirect and mediated by adaptor proteins, for example, Rab GTPases. Rab27a, via effector melanophilin (Mlph), recruits myosin-Va (MyoVa) to melanosomes and thereby disperses them into melanocyte dendrites. To better understand how adaptors regulate motor:cargo interaction, we used single melanosome fluorescence recovery after photobleaching (smFRAP) to characterize the association kinetics among MyoVa, its adaptors, and melanosomes. We found that MyoVa and Mlph rapidly recovered after smFRAP, whereas Rab27a did not, indicating that MyoVa and Mlph dynamically associate with melanosomes and Rab27a does not. This suggests that dynamic Rab27a:effector interaction rather than Rab27a melanosome:cytosol cycling regulates MyoVa:melanosome association. Accordingly, a Mlph-Rab27a fusion protein reduced MyoVa smFRAP, indicating that it stabilized melanosomal MyoVa. Finally, we tested the functional importance of dynamic MyoVa:melanosome interaction. We found that whereas a MyoVa-Rab27a fusion protein dispersed melanosomes in MyoVa-deficient cells, dendrites were significantly less elongated than in wild-type cells. Given that dendrites are the prime sites of melanosome transfer from melanocytes to keratinocytes, we suggest that dynamic MyoVa:melanosome interaction is important for pigmentation in vivo.