Constitutive formation of caveolae in a bacterium.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Liver Lipoma in a Dog: Case Report and Literature Review.

Lipoma of the liver has not been reported in dogs. An 8 yr old spayed female Great Dane was referred for diagnostic workup of abdominal distention. Computed tomography showed fat-attenuating masses with negative attenuation values (variable between -60 to -40 Hounsfield units) and minimal contrast uptake within the left cranial abdomen. Left lateral and right medial liver lobectomies were performed to remove two liver masses. Histopathology showed large lipomas arising from within the hepatic parenchyma. Immunohistochemistry for smooth muscle actin was negative, consistent with true lipomas. The dog was euthanized 8 mo later because of causes likely unrelated to the liver lipoma. This is the first case report of lipoma in the liver of a dog. The purpose of this case report and brief literature review is to provide evidence that surgical excision of fat-attenuating masses within the liver that are consistent with lipoma using immunohistochemistry can be curative.

Detection of intra-cardiac thrombi and congestive heart failure in cats using computed tomographic angiography.

Arterial thromboembolism is a life-threatening condition in cats most commonly secondary to cardiac disease. Echocardiography is the reference standard to evaluate for presence of a thrombus. In humans, computed tomographic (CT) angiography is becoming widely used to detect left atrial thrombi precluding the use of sedation. The purpose of this prospective, controlled, methods comparison pilot study was threefold: (1) describe new CT angiography protocol used in awake cats with cardiac disease and congestive heart failure; (2) determine accuracy of continuous and dynamic acquisition CT angiography to identify and characterize cardiac thrombi from spontaneous echocardiographic contrast using transthoracic echocardiography as our reference standard; (3) identify known negative prognostic factors and comorbidities of the thorax that CT angiography may provide that complement or supersede echocardiographic examination. Fourteen cats with heart disease were recruited; 7 with thrombi and 7 with spontaneous echocardiographic contrast. Echocardiography and awake CT angiography were performed using a microdose of contrast. Six of 7 thrombi were identified on CT angiography as filling defects by at least one reviewer within the left auricle (n = 6) and right heart (n = 1). Highest sensitivity (71.4%) was in continuous phase and highest specificity (85.7%) was in dynamic studies with fair to moderate interobserver agreement (0.38 and 0.44). CT angiography identified prognostic cardiac information (left atrial enlargement, congestive heart failure, arterial thromboembolism) and comorbidities (suspected idiopathic pulmonary fibrosis, asthma). This study indicates CT angiography can readily identify cardiac thrombi, important prognostic information and comorbidities, and can be safely performed in cats with cardiac disease and congestive heart failure.

Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis.

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.

SYSTEMIC BLASTOMYCOSIS IN A CAPTIVE RED RUFFED LEMUR (VARECIA RUBRA).

A 5-yr-old, intact male red ruffed lemur ( Varecia rubra ) presented for evaluation as the result of a 1-wk history of lethargy and hyporexia. Physical examination findings included thin body condition, muffled heart sounds, harsh lung sounds, and liquid brown diarrhea. Complete blood count and serum biochemistry showed an inflammatory leukogram, mild hyponatremia, and mild hypochloremia. Orthogonal trunk radiographs revealed a severe alveolar pattern in the right cranial lung lobes with cardiac silhouette effacement. Thoracic ultrasound confirmed a large, hypoechoic mass in the right lung lobes. Fine-needle aspiration of the lung mass and cytology revealed fungal yeast organisms, consistent with Blastomyces dermatitidis. Blastomyces Quantitative EIA Test on urine was positive. Postmortem examination confirmed systemic blastomycosis involving the lung, tracheobronchial lymph nodes, spleen, kidney, liver, cerebrum, and eye. To the authors' knowledge, this is the first report of blastomycosis in a prosimian species.

Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

Redistribution of caveolae during mitosis.

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis.

The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway.

Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

Evaluation of bronchial narrowing in coughing dogs with heart murmurs using computed tomography.

BACKGROUND: The origin of cough in dogs with heart murmurs is controversial, because the cough could be primary cardiac (eg, pulmonary edema, bronchi compression by left-sided cardiomegaly) or respiratory (eg, bronchomalacia, other bronchial or bronchiolar disease, interstitial lung disease) in origin. HYPOTHESIS/OBJECTIVES: To study the association between left atrium (LA) dilatation and cardiomegaly and bronchial narrowing in coughing dogs with heart murmurs using computed tomography (CT). ANIMALS: Twenty-one client-owned coughing dogs with heart murmurs and 14 historical control dogs. METHODS: Dogs with cough and murmur were prospectively recruited over 4 months. Cervical and thoracic radiography, echocardiography, and thoracic CT were performed in enrolled dogs. Control dogs, with no disease on thoracic CT and no records of heart murmur and coughing, were gathered from the institution's computerized database. Degree of bronchial narrowing was assessed using the bronchial-to-aorta (Ao) ratio, measured by 3 radiologists blinded to the clinical findings. After identifying bronchi that were significantly narrowed in dogs with murmur compared to controls, the relationship between degree of narrowing and LA/Ao ratio (measured echocardiographically) and vertebral heart scale (VHS) measured radiographically was studied in dogs with murmur using mixed-effects regression. RESULT: Significant narrowing was identified for all left-sided bronchi and the right principal, middle, and caudal bronchi in the coughing dogs, compared with controls. Increasing LA size and VHS were significantly inversely associated with diameter for all left-sided and right-sided bronchi indicated above. CONCLUSION AND CLINICAL IMPORTANCE: Results indicate an association between LA enlargement and cardiomegaly and bronchial narrowing and support heart size-associated exacerbation of cough in dogs with murmurs.

The post-translational modification landscape of commercial beers.

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

Correction: Population Distribution Analyses Reveal a Hierarchy of Molecular Players Underlying Parallel Endocytic Pathways.

[This corrects the article DOI: 10.1371/journal.pone.0100554.].

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers.

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of ß1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.

The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments.

The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.

EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization.

Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading.

The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

Revisiting caveolin trafficking: the end of the caveosome.

In this issue, a study by Hayer et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003086) provides insights into the trafficking of caveolins, the major membrane proteins of caveolae. As well as providing evidence for ubiquitin-mediated endosomal sorting and degradation of caveolin in multivesicular bodies (MVBs), the new findings question the existence of a unique organelle proposed nine years ago, the caveosome.

Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis.

Eukaryotic cells deftly coordinate an array of endocytic pathways beyond the classical clathrin-mediated endocytic route. Although the existence of clathrin-independent endocytic pathways has been accepted for some time, only recently have tools been developed that specifically delineate their fine details, including molecular composition and ultrastructural morphology. Identification of the salient features of distinct pathways has concomitantly attributed them with specific roles during important cellular processes. Insight from model organisms confirms these roles and suggests maintenance of crucially adapted functions across species. Among other roles, clathrin-independent endocytosis has now been linked to plasma membrane repair, cellular spreading, cellular polarization, and modulation of intercellular signaling. The field is now primed to identify how these pathways function within physiologically relevant environments.

Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells.

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.