Michele Bastiani

PTRF-Cavin, a Conserved Cytoplasmic Protein Required for Caveola Formation and Function

Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.

Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

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.

Caveolae at a glance

The plasma membrane is more than a simple delimitation of the boundary of the cell but is a dynamic multi-domain membrane system participating in numerous cellular processes. In many different cell types, the plasma membrane is heavily decorated with small pits of 60–80 nm in diameter, which

Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae.

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae. DOI: http://dx.doi.org/10.7554/eLife.01434.001

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Phosphocaveolin-1 regulates a positive feedback loop that responds to mechanical stress to induce caveola biogenesis by relieving Egr1 transcriptional inhibition of caveolin-1 and cavin-1.

PTRF-cavin -1 expression decreases the migration of PC3 prostate cancer cells: Role of matrix metalloprotease 9

Caveolae are specialized plasma membrane subdomains with a distinct lipid and protein composition, which play an essential role in cell physiology by performing trafficking and signalling functions. The structure and functions of caveolae have been shown to require caveolin-1, a major protein component of caveolae. Caveolin-1 expression and secretion are increased in metastatic prostate cancer, and caveolin-1 seems to contribute to prostate cancer growth and metastasis. Recently, a cytoplasmic protein named PTRF (Polymerase I and Transcript Release Factor) or cavin-1 was found to be required, in concert with caveolin-1, for the formation and functions of caveolae. Genetic ablation of PTRF results in loss of caveolae while caveolin-1 is still expressed, albeit at reduced level, but associates with flat plasma membrane. In metastatic PC3 prostate cancer cells that express abundant caveolin-1 but no PTRF, heterologous PTRF expression restores caveola formation and caveolin-1 distribution (Hill et al., 2008; Cell 132, 113-124). We now show that PTRF/cavin-1-expressing PC3 cells exhibit decreased migration, and that this effect is mediated by reduced MMP9 production. PTRF/cavin-1, and to a lesser extent, cavin-2, -3, and -4 all decreased MMP9. We further show that the PTRF/cavin-1-mediated reduction of MMP9 production is independent of caveola formation. Taken together, our results suggest that PTRF/cavin-1 expression alters prostate cancer aggressiveness.

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

The caveolar membrane microdomain plays an integral role in stabilizing the muscle fiber surface in mice and zebrafish.

Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.

Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway.

Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative‐dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial‐dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase‐3/7 activity. Retinol induced oxidant‐ and time‐dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase‐3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co‐administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role.