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None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β(2)-adrenoreceptor (β(2)-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β(2)-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β(2)-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.

SummaryThe cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems: mouse and zebrafish. In both models Cavin4 localized to T-tubules and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicatedcavin4paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein, Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

Description, RyR1 Ca2+ leak causes a cascade of events that shifts Ca2+ to the cytoplasm and mitochondria, supporting force generation., Muscle contraction depends on tightly regulated Ca2+ release. Aberrant Ca2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.

Identification of protein interaction networks is key for understanding intricate biological processes, but mapping such networks is challenging with conventional biochemical methods, especially for weak or transient interactions. Proximity-dependent biotin labelling (BioID) using promiscuous biotin ligases and mass spectrometry (MS)-based proteomics has emerged in the past decade as a powerful method for probing local proteomes and protein interactors. Here, we describe the application of an engineered biotin ligase, TurboID, for proteomic mapping and interactor screening in vivo in zebrafish. We generated novel transgenic zebrafish lines that express TurboID fused to a conditionally stabilised GFP-binding nanobody, dGBP, which targets TurboID to the GFP-tagged proteins of interest. The TurboID-dGBP zebrafish lines enable proximity-dependent biotin labelling in live zebrafish simply through outcrossing with existing GFP-tagged lines. Here, we outline a detailed protocol of the BLITZ method (Biotin Labelling In Tagged Zebrafish) for utilising TurboID-dGBP fish lines to map local proteomes and screen novel interactors. Graphic abstract: [Image: see text] Schematic overview of the BLITZ method. TurboID-dGBP fish are crossed with GFP-tagged lines to obtain embryos co-expressing TurboID-dGBP (indicated by mKate2) and the GFP-POI (protein of interest). Embryos expressing only TurboID are used as a negative control. Embryos (2 to 7 dpf) are incubated overnight with a 500 μM biotin-supplemented embryo medium. This biotin incubation step allows TurboID to catalyse proximity-dependent biotinylation in live zebrafish embryos. After biotin incubation, embryos are solubilised in lysis buffer, and free biotin is removed using a PD-10 desalting column. The biotinylated proteins are captured by streptavidin affinity purification, and captured proteins are analysed by MS sequencing.

Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

Caveolae-associated protein 3 (cavin3), a putative tumor suppressor protein, is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays, we show a direct interaction between BRCA1 and cavin3. Association of BRCA1 and cavin3 occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Supporting a role in DNA repair, cavin3-deficient cells were sensitized to the effects of PARP inhibition, which compromises DNA repair, and showed reduced recruitment of the BRCA1 A-complex to UV DNA damage foci. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. We conclude that cavin3 functions together with BRCA1 in multiple pathways that contribute to tumorigenesis.

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex., It is unclear how the T-tubule structure of skeletal muscle, which regulates coordinated muscle contraction, forms. Here, the authors develop a four-dimensional quantitative model for T-tubule formation in zebrafish, based on live imaging, proposing a dynamic endocytic capture model.

The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction that shows dysmorphology in a number of genetically inherited muscle diseases. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we have used the optically transparent and genetically tractable zebrafish system to probe T-tubule developmentin vivo. By combining live imaging with three-dimensional electron microscopy we derived a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formationin vivowe developed a quantitative screen for proteins that associate with and modulate early T-tubule formation including an overexpression screen of the entire zebrafish Rab protein family. We propose a new endocytic capture model involving i) formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma ii) stabilization by myofibrils/sarcoplasmic reticulum and iii) delivery of membrane from the recycling endosome and Golgi complex.

The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

Caveolae, small bulb-like pits, are the most abundant surface feature of many vertebrate cell types. The relationship of the structure of caveolae to their function has been a subject of considerable scientific interest in view of the association of caveolar dysfunction with human disease. In a recent study Lo et al.1 investigated the organization and function of caveolae in skeletal muscle. Using quantitative 3D electron microscopy caveolae were shown to be predominantly organized into multilobed structures which provide a large reservoir of surface-connected membrane underlying the sarcolemma. These structures were preferentially disassembled in response to changes in membrane tension. Perturbation or loss of caveolae in mouse and zebrafish models suggested that caveolae can protect the muscle sarcolemma against damage in response to excessive membrane activity. Flattening of caveolae to release membrane into the bulk plasma membrane in response to increased membrane tension can allow cell shape...

Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.

SummaryCaveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1−/− mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1−/− mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.

The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic “skeleton” is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b −/− zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b −/− mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechanoprotection. Refereed/Peer-reviewed

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

Recent studies have identified a number of forms of muscular dystrophy, termed dystroglycanopathies, which are associated with loss of natively glycosylated alpha-dystroglycan. Here we identify a new animal model for this class of disorders in Sphynx and Devon Rex cats. Affected cats displayed a slowly progressive myopathy with clinical and histologic hallmarks of muscular dystrophy including skeletal muscle weakness with no involvement of peripheral nerves or CNS. Skeletal muscles had myopathic features and reduced expression of alpha-dystroglycan, while beta-dystroglycan, sarcoglycans, and dystrophin were expressed at normal levels. In the Sphynx cat, analysis of laminin and lectin binding capacity demonstrated no loss in overall glycosylation or ligand binding for the alpha-dystroglycan protein, only a loss of protein expression. A reduction in laminin-alpha2 expression in the basal lamina surrounding skeletal myofibers was also observed. Sequence analysis of translated regions of the feline dystroglycan gene (DAG1) in affected cats did not identify a causative mutation, and levels of DAG1 mRNA determined by real-time QRT-PCR did not differ significantly from normal controls. Reduction in the levels of glycosylated alpha-dystroglycan by immunoblot was also identified in an affected Devon Rex cat. These data suggest that muscular dystrophy in Sphynx and Devon Rex cats results from a deficiency in alpha-dystroglycan protein expression, and as such may represent a new type of dystroglycanopathy where expression, but not glycosylation, is affected.

Transmembrane water transport is mediated by aquaporins (AQPs), of which AQP1 and AQP4 are expressed in skeletal muscle. AQP4 expression is reduced in Duchenne muscular dystrophy (DMD) patients, and is reported to correlate with decreased alpha1-syntrophin and altered osmotic permeability. In this study, we assessed the relationship between AQP1, AQP4, dystrophin and alpha1-syntrophin in dystrophinopathy and dysferlinopathy patients. Muscle biopsies of patients with DMD (n = 8) and limb-girdle muscular dystrophy type 2B (LGMD2B; n = 5) were screened for AQP1 and AQP4 expression by real-time quantitative RT-PCR or Western blot and immunohistochemistry. AQP expression was further analyzed in primary myotubes derived from DMD and LGMD2B patients by cell culture and immunohistochemistry. AQP1 transcript and protein expression was significantly elevated in DMD biopsies, and was localized to the sarcolemma of muscle fibers and endothelia of muscle capillaries. AQP4 was significantly reduced despite normal dystrophin and alpha1-syntrophin in dysferlinopathy patients, while expression of AQP1 was variably upregulated. Expression of AQP1 and AQP4 was normal in patient-derived primary myotubes, suggesting that altered AQPs observed in biopsies are likely secondary to the dystrophic process. Our study shows that AQP4 downregulation can occur in muscular dystrophies with either normal or disrupted expression of dystrophin-associated proteins, and that this might be associated with upregulation of AQP1.

The objective of this study is to expand the applications of MyoD-forced myogenesis for research and diagnosis of human muscle disorders using a lentiviral vector (LVhMyoD) for efficient trans-differentiation of patient primary cells. LVhMyoD transduced cells readily formed striated, multinucleate myotubes expressing a wide range of genes associated with muscular dystrophy (dystrophin, dysferlin, sarcoglycans, caveolin-3) and congenital myopathy (nebulin, actin, desmin, tropomyosin, troponin). We demonstrate that MyoD gene-modified fibroblasts reproduce protein deficiencies associated with different forms of muscular dystrophy, and confirm that LVhMyoD gene-modified chorionic villus can be used successfully to determine the dystrophin status of the developing fetus, augmenting prenatal diagnosis of dystrophinopathy patients. Using muscle-specific cDNA derived from LVhMyoD gene-modified patient cells, we identified a female carrier bearing a large dystrophin deletion and a previously unidentified non-coding splice-site mutation within dystrophin in a Becker muscular dystrophy patient. This study highlights the significant potential of lentiviral MyoD-forced myogenesis for study of a wide range of human muscle disorders; a field constrained by the limited availability of human tissue. LVhMyoD gene-modified patient cells provide a renewable source of mutant protein and muscle-specific mRNA, facilitating accelerated mutation screening of large genes, molecular analyses of splicing abnormalities and study of disease-causing mutations.

A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ∼500–600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ∼95 nm in width with ∼60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.

A case of rippling muscle disease is presented and features of this rare condition, and its association with caveolin-3 are discussed.

Protein modification with isoprenoid lipids affects hundreds of signaling proteins in eukaryotic cells. Modification of isoprenoids with reporter groups is the main approach for the creation of probes for the analysis of protein prenylation in vitro and in vivo. Here, we describe a new strategy for the synthesis of functionalized phosphoisoprenoids that uses an aminederivatized isoprenoid scaffold as a starting point for the synthesis of functionalized phosphoisoprenoid libraries. This overcomes a long-standing problem in the field, where multistep synthesis had to be carried out for each individual isoprenoid analogue. The described approach enabled us to synthesize a range of new compounds, including two novel fluorescent isoprenoids that previously could not be generated by conventional means. The fluorescent probes that were developed using the described approach possess significant spectroscopic advantages to all previously generated fluorescent isoprenoid analogue. Using these analogues for flow cytometry and cell imaging, we analyzed the uptake of isoprenoids by mammalian cells and zebrafish embryos. Furthermore, we demonstrate that derivatization of the scaffold can be coupled in a one-pot reaction to enzymatic incorporation of the resulting isoprenoid group into proteins. This enables rapid evaluation of functional groups for compatibility with individual prenyltransferases and identification of the prenyltransferase specific substrates.

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.

We characterized the frequency of limb–girdle muscular dystrophy (LGMD) subtypes in a cohort of 76 Australian muscular dystrophy patients using protein and DNA sequence analysis. Calpainopathies (8%) and dysferlinopathies (5%) are the most common causes of LGMD in Australia. In contrast to European populations, cases of LGMD2I (due to mutations in FKRP) are rare in Australasia (3%). We have identified a cohort of patients in whom all common disease candidates have been excluded, providing a valuable resource for identification of new disease genes. Cytoplasmic localization of dysferlin correlates with fiber regeneration in a subset of muscular dystrophy patients. In addition, we have identified a group of patients with unidentified forms of LGMD and with markedly abnormal dysferlin localization that does not correlate with fiber regeneration. This pattern is mimicked in primary caveolinopathy, suggesting a subset of these patients may also possess mutations within proteins required for membrane targeting of dysferlin.

Aquaporins (AQPs) are a family of water channel proteins that assist in maintenance of the cellular osmotic environment and whole body fluid balance. Specialized organ-specific AQPs are important in physiologic and pathologic processes but little is known about AQPs in the human heart. AQP1 has been identified in rodent heart. We investigated the presence and localization of AQP1 in human heart and skeletal muscle using immunohistochemistry and confocal microscopy, western blot and reverse transcriptase-polymerase chain reaction. There was abundant AQP1 present in both cardiac and skeletal muscle. Immunohistochemistry revealed co-localization of AQP1 with vinculin, a t-tubule marker, and caveolin-3. No novel sequences bearing an NPA box motif common to other AQPs were identified in human heart using degenerative PCR analysis. We conclude that AQP1 is present in the human heart. AQP1 co-localizes with t-tubular and caveolar proteins. Cardiac AQPs may have a role during osmotic stresses including ischemia/reperfusion and cardiopulmonary bypass.

Caveolar proteins and caveolae negatively regulate a second clathrin-independent endocytic CLIC/GEEC pathway; caveolin-1 affects membrane diffusion properties of raft-associated CLIC cargo, and the scaffolding domain of caveolin-1 is required and sufficient for endocytic inhibition., Several studies have suggested crosstalk between different clathrin-independent endocytic pathways. However, the molecular mechanisms and functional relevance of these interactions are unclear. Caveolins and cavins are crucial components of caveolae, specialized microdomains that also constitute an endocytic route. Here we show that specific caveolar proteins are independently acting negative regulators of clathrin-independent endocytosis. Cavin-1 and Cavin-3, but not Cavin-2 or Cavin-4, are potent inhibitors of the clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, in a process independent of caveola formation. Caveolin-1 (CAV1) and CAV3 also inhibit the CLIC/GEEC pathway upon over-expression. Expression of caveolar protein leads to reduction in formation of early CLIC/GEEC carriers, as detected by quantitative electron microscopy analysis. Furthermore, the CLIC/GEEC pathway is upregulated in cells lacking CAV1/Cavin-1 or with reduced expression of Cavin-1 and Cavin-3. Inhibition by caveolins can be mimicked by the isolated caveolin scaffolding domain and is associated with perturbed diffusion of lipid microdomain components, as revealed by fluorescence recovery after photobleaching (FRAP) studies. In the absence of cavins (and caveolae) CAV1 is itself endocytosed preferentially through the CLIC/GEEC pathway, but the pathway loses polarization and sorting attributes with consequences for membrane dynamics and endocytic polarization in migrating cells and adult muscle tissue. We also found that noncaveolar Cavin-1 can act as a modulator for the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. This work provides new insights into the regulation of noncaveolar clathrin-independent endocytosis by specific caveolar proteins, illustrating multiple levels of crosstalk between these pathways. We show for the first time a role for specific cavins in regulating the CLIC/GEEC pathway, provide a new tool to study this pathway, identify caveola-independent functions of the cavins and propose a novel mechanism for inhibition of the CLIC/GEEC pathway by caveolin., Author Summary Endocytosis is the process that allows cells to take up molecules from the environment. Several endocytic pathways exist in mammalian cells. While the best understood endocytic pathway uses clathrin, recent years have seen a great increase in our understanding of clathrin-independent endocytic pathways. Here we characterize the crosstalk between caveolae, flask-shaped specialized microdomains present at the plasma membrane, and a second clathrin-independent pathway, the CLIC/GEEC Cdc42-regulated endocytic pathway. These pathways are segregated in migrating cells with caveolae at the rear and CLIC/GEEC endocytosis at the leading edge. Here we find that specific caveolar proteins, caveolins and cavins, can also negatively regulate the CLIC/GEEC pathway. With the help of several techniques, including quantitative electron microscopy analysis and real-time live-cell imaging, we demonstrate that expression of caveolar proteins affects early carrier formation, causes cellular lipid changes, and changes the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. The functional consequences of loss of caveolar proteins on the CLIC/GEEC pathway included inhibition of polarized cell migration and increased endocytosis in tissue explants.

Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts. Dysferlin shows only partial overlap with Cav-3 on the surface of isolated muscle fibers but co-localizes with Cav-3 in developing transverse (T)-tubules in muscle cell lines. Heterologously expressed dystrophy-associated mutant Cav3R26Q accumulates in the Golgi complex of muscle cell lines or fibroblasts. Cav3R26Q and other Golgi-associated mutants of both Cav-3 (Cav3P104L) and Cav-1 (Cav1P132L) caused a dramatic redistribution of dysferlin to the Golgi complex. Heterologously expressed epitope-tagged dysferlin associates with the plasma membrane in primary fibroblasts and muscle cells. Transport to the cell surface is impaired in the absence of Cav-1 or Cav-3 showing that caveolins are essential for dysferlin association with the PM. These results suggest a functional role for caveolins in a novel post-Golgi trafficking pathway followed by dysferlin.