Cell surface plasticity in response to shape change in the whole organism.

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

Caveolae sense oxidative stress through membrane lipid peroxidation and cytosolic release of CAVIN1 to regulate NRF2.

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 organelle in cellular homeostasis and wound response.

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle.

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.

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.

Caveolae-associated protein 3 (cavin3) 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 revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.

When cells become cancerous they often stop making certain proteins. This includes a protein known as cavin3 which resides in bulb-shaped pits of the membrane that surrounds the cell called caveolae. These structures work like stress detectors, picking up changes in the membrane and releasing proteins, such as cavin3, into the cell's interior. Past studies suggest that cavin3 might interact with a protein called BRCA1 that suppresses the formation of tumors. Cells with mutations in the gene for BRCA1 struggle to fix damage in their DNA, and have to rely on other repair proteins, such as PARPs (short for poly (ADP-ribose) polymerases). Blocking PARP proteins with drugs can kill cancer cells with problems in their BRCA1 proteins. However, it was unclear what role cavin3 plays in this mechanism. To investigate this, McMahon et al. exposed cells grown in the laboratory to DNA-damaging UV light to stimulate the release of cavin3 from caveolae. This revealed that cavin3 interacts with BRCA1 when cells are under stress, and helps stabilize the protein so it can perform DNA repairs. Cells without cavin3 showed decreased levels of the BRCA1 protein, but compensated for the loss of BRCA1 by increasing the levels of their PARP proteins. These cells also had increased DNA damage following treatment with drugs that block PARPs, similar to cancer cells carrying mutations in the gene for BRCA1. These findings suggest that cavin3 helps BRCA1 to suppress the formation of tumors, and therefore should be considered when developing new anti-cancer treatments.

A robust method for particulate detection of a genetic tag for 3D electron microscopy.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish.

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.

Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.

In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation.

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.

Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis.

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Gomesin peptides prevent proliferation and lead to the cell death of devil facial tumour disease cells.

The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease.

Caveolae Protect Notochord Cells against Catastrophic Mechanical Failure during Development.

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.

A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity.

Caveolae and associated cavin and caveolins may govern myocardial function, together with responses to mechanical and ischaemic stresses. Abnormalities in these proteins are also implicated in different cardiovascular disorders. However, specific roles of the cavin-1 protein in cardiac and coronary responses to mechanical/metabolic perturbation remain unclear. We characterised cardiovascular impacts of cavin-1 deficiency, comparing myocardial and coronary phenotypes and responses to stretch and ischaemia-reperfusion in hearts from cavin-1 +/+ and cavin-1 -/- mice. Caveolae and caveolins 1 and 3 were depleted in cavin-1 -/- hearts. Cardiac ejection properties in situ were modestly reduced in cavin-1 -/- mice. While peak contractile performance in ex vivo myocardium from cavin-1 -/- and cavin-1 +/+ mice was comparable, intrinsic beating rate, diastolic stiffness and Frank-Starling behaviour (stretch-dependent diastolic and systolic forces) were exaggerated in cavin-1 -/- hearts. Increases in stretch-dependent forces were countered by NOS inhibition (100 µM L-NAME), which exposed negative inotropy in cavin-1 -/- hearts, and were mimicked by 100 µM nitroprusside. In contrast, chronotropic differences appeared largely NOS-independent. Cavin-1 deletion also induced NOS-dependent coronary dilatation, ≥3-fold prolongation of reactive hyperaemic responses, and exaggerated pressure-dependence of coronary flow. Stretch-dependent efflux of lactate dehydrogenase and cardiac troponin I was increased and induction of brain natriuretic peptide and c-Fos inhibited in cavin-1 -/- hearts, while ERK1/2 phospho-activation was preserved. Post-ischaemic dysfunction and damage was also exaggerated in cavin-1 -/- hearts. Diverse effects of cavin-1 deletion reveal important roles in both NOS-dependent and -independent control of cardiac and coronary functions, together with governing sarcolemmal fragility and myocardial responses to stretch and ischaemia.

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.

The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle.

Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1(-/-) muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin-cavin system.

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.

Caveolin-1 is necessary for hepatic oxidative lipid metabolism: evidence for crosstalk between caveolin-1 and bile acid signaling.

Caveolae 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.

Ski-interacting protein (SKIP) interacts with androgen receptor in the nucleus and modulates androgen-dependent transcription.

BACKGROUND: The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-inducible DNA transcription factors, and is the major mediator of male sexual development, prostate growth and the pathogenesis of prostate cancer. Cell and gene specific regulation by the AR is determined by availability of and interaction with sets of key accessory cofactors. Ski-interacting protein (SKIP; SNW1, NCOA62) is a cofactor shown to interact with several NRs and a diverse range of other transcription factors. Interestingly, SKIP as part of the spliceosome is thought to link mRNA splicing with transcription. SKIP has not been previously shown to interact with the AR. RESULTS: The aim of this study was to investigate whether SKIP interacts with the AR and modulates AR-dependent transcription. Here, we show by co-immunoprecipitation experiments that SKIP is in a complex with the AR. Moreover, SKIP increased 5α-dihydrotestosterone (DHT) induced N-terminal/C-terminal AR interaction from 12-fold to almost 300-fold in a two-hybrid assay, and enhanced AR ligand-independent AF-1 transactivation. SKIP augmented ligand- and AR-dependent transactivation in PC3 prostate cancer cells. Live-cell imaging revealed a fast (half-time=129 s) translocation of AR from the cytoplasm to the nucleus upon DHT-stimulation. Förster resonance energy transfer (FRET) experiments suggest a direct AR-SKIP interaction in the nucleus upon translocation. CONCLUSIONS: Our results suggest that SKIP interacts with AR in the nucleus and enhances AR-dependent transactivation and N/C-interaction supporting a role for SKIP as an AR co-factor.