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The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attentiondeficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or “endocytic brake,” controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.

Aims/hypothesis During pregnancy, adipose tissue (AT) must expand to support the growing fetus and the future nutritional needs of the offspring. Limited expandability of AT is associated with insulin resistance, attributed to ectopic lipid deposition. This study aimed to investigate human AT expandability during pregnancy and its role in the pathogenesis of gestational diabetes mellitus (GDM). Methods This cross-sectional study of omental (OM) and subcutaneous (SQ) AT collected at Caesarean delivery included 11 pregnant and three non-pregnant women with normal glucose tolerance (NGT), five with GDM, three with type 2 diabetes mellitus. Adipocyte size, capillary density, collagen content and capillary growth were measured. Affymetrix arrays and real-time PCR studies of gene expression were performed. Results Mean OM adipocyte size was greater in women with GDM than in those with NGT ( p  = 0.004). Mean OM and SQ capillary density was lower in GDM compared with NGT ( p  = 0.015). Capillary growth did not differ significantly between groups. The most differentially expressed AT transcript when comparing non-pregnant and pregnant women corresponded to the IGF binding protein (IGFBP)-5, the expression levels of which was found by subsequent quantitative real-time PCR to be lower in women with GDM vs women with NGT ( p  < 0.0001). Conclusions/interpretation The relative OM adipocyte hypertrophy and decreased OM and SQ capillary density are consistent with impaired AT expandability in GDM. The induction of adipose tissue IGFBP5 in pregnancy and its decrease in GDM point to the importance of the IGF-1 signalling pathway in AT expansion in pregnancy and GDM susceptibility.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.Micro-Meta App is an intuitive, highly interoperable, open-source software tool designed to facilitate the extraction and collection of relevant microscopy metadata as specified by recent community guidelines.

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl − channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence. TMEM16A acts in smooth muscle cells as a critical pacemaker channel that enables the internal anal sphincter to contract rhythmically and continuously, a critical phenomenon to allow for fecal continence.

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca2+ channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca2+ release (VICaR) from internal Ca2+ stores without Ca2+ influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1I4895T/WT (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca2+ influx elicited Ca2+syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca2+, IT/+ terminals showed a twofold decrease in global Ca2+ transients, with no change in plasmalemmal Ca2+ current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na +/Ca 2+ exchanger, the predominant Na + channel isoform rH1, and the L-type calcium channel, Ca v1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na +/Ca 2+ exchanger or the Na + channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in ≈30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-β receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans, which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.

Ion channels in myometrial cells play critical roles in spontaneous and agonist-induced uterine contraction during the menstrual cycle, pregnancy maintenance, and parturition; thus, identifying the genes of ion channels in these cells and determining their roles are essential to understanding the biology of reproduction. Previous studies with in vitro functional and pharmacological approaches have produced controversial results regarding the presence and role of TMEM16A Ca2+-activated Cl– channels in myometrial cells. To unambiguously determine the function of this channel in these cells, we employed a genetic approach by using smooth muscle cell-specific TMEM16A deletion (i.e. TMEM16ASMKO) mice. We found that myometrial cells from TMEM16ASMKO mice generated the same pattern and magnitude in Ca2+ signals upon stimulation with KCl, oxytocin, and PGF2α compared to the isogenic control myometrial cells. At the uterine tissue level, TMEM16A deletion also did not cause detectable changes in either spontaneous or agonist (i.e. KCl, oxytocin, and PGF2α)-induced contractions. Moreover, in vivo the TMEM16ASMKO mice gave birth at full term with the same litter size as genetically identical control mice. Finally, TMEM16A immunostaining in both control and TMEM16ASMKO mice revealed that this protein was highly expressed in the endometrial stroma, but did not co-localize with a smooth muscle specific marker MYH11. Collectively, these results unequivocally demonstrate that TMEM16A does not serve as a pacemaking channel for spontaneous uterine contraction, neither does it function as a depolarizing channel for agonist-evoked uterine contraction. Yet these two functions could underlie the normal gestation length and litter size in the TMEM16ASMKO mice. Summary Sentence The TMEM16A is absent in myometrial cells and exerts no impact on Ca2+ signaling, contractile responses and pregnancy in mice.