Antibody-specific detection of caveolin-1 in subapical compartments of MDCK cells

Caveolin-1 is the major structural component of caveolae and is also found in the Golgi complex of many cell types. Occasionally, caveolin-1 has been observed in additional intracellular compartments, including recycling endosomes. Why caveolin-1 expression is detected at these sites only infrequently is not clear. In this study, we test the hypothesis that non-caveolar, non-Golgi pools of caveolin-1 display unique and/or fixation-dependent epitopes. We compared the ability of a panel of antibodies raised against various domains of caveolin-1 to detect distinct subcellular pools of the protein by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells, a cell line where the subcellular localization of caveolin-1 has been extensively characterized. We show that three antibodies directed to the N-terminus of caveolin-1 recognize a previously undetected pool of caveolin-1 in the subapical region of MDCK cells, a localization characteristic of endosomal recycling compartments. The antibodies vary in their ability to label caveolin-1 at the cell surface, and the epitopes detected by each are highly fixation dependent. Our findings suggest that no single caveolin antibody or staining condition is capable of detecting all the caveolin-1 in a cell simultaneously. Consequently, the subcellular distribution of caveolin-1 may be much broader than currently believed.     

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.     

Presenilin-1-mediated retention of APP derivatives in early biosynthetic compartments

Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin-1 (PS-1) plays a key role in the final step of Abeta formation, the gamma-secretase cleavage. Previously, we showed that PS-1 is retained in pre-Golgi compartments by incorporation into COPI-coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS-1 also mediates the retention of the beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta in VTCs as well as their distribution to COPI-coated VTC membranes. By contrast, overexpression of the dominant-negative aspartate mutant PS-1(D257A) or PS-knockout decreased incorporation of these APP derivatives into COPI-coated membranes. Sorting of APP derivatives to COPI-coated VTC membranes was not depending on the APP cytosolic tail. In post-Golgi compartments, PS-1 expression enhanced the association of full-length APP/APPs with endosomal compartments at the expense of plasma membrane-bound APP. We conclude that PS-1, in addition to its role in gamma-secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS-1 in this role may have important consequences for the progress of AD.     

Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments

Many fungal parasites enter plant cells by penetrating the host cell wall and, thereafter, differentiate specialized intracellular feeding structures, called haustoria, by invagination of the plant's plasma membrane. Arabidopsis PEN gene products are known to act at the cell periphery and function in the execution of apoplastic immune responses to limit fungal entry. This response underneath fungal contact sites is tightly linked with the deposition of plant cell wall polymers, including PMR4/GSL5-dependent callose, in the paramural space, thereby producing localized wall thickenings called papillae. We show that powdery mildew fungi specifically induce the extracellular transport and entrapment of the fusion protein GFP–PEN1 syntaxin and its interacting partner monomeric yellow fluorescent protein (mYFP)–SNAP33 within the papillary matrix. Remarkably, PMR4/GSL5 callose, GFP–PEN1, mYFP–SNAP33, and the ABC transporter GFP–PEN3 are selectively incorporated into extracellular encasements surrounding haustoria of the powdery mildew Golovinomyces orontii , suggesting that the same secretory defense responses become activated during the formation of papillae and haustorial encasements. This is consistent with a time-course analysis of the encasement process, indicating that these extracellular structures are generated through the extension of papillae. We show that PMR4/GSL5 callose accumulation in papillae and haustorial encasements occurs independently of PEN1 syntaxin. We propose a model in which exosome biogenesis/release serves as a common transport mechanism by which the proteins PEN1 and PEN3, otherwise resident in the plasma membrane, together with membrane lipids, become stably incorporated into both pathogen-induced cell wall compartments.     

High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport

The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.     

PI4P and Rab inputs collaborate in myosin-V-dependent transport of secretory compartments in yeast

Cell polarity involves transport of specific membranes and macromolecules at the right time to the right place. In budding yeast, secretory vesicles are transported by the myosin-V Myo2p to sites of cell growth. We show that phosphatidylinositol 4-phosphate (PI4P) is present in late secretory compartments and is critical for their association with, and transport by, Myo2p. Further, the trans-Golgi network Rab Ypt31/32p and secretory vesicle Rab Sec4p each bind directly, but distinctly, to Myo2p, and these interactions are also required for secretory compartment transport. Enhancing the interaction of Myo2p with PI4P bypasses the requirement for interaction with Ypt31/32p and Sec4p. Together with additional genetic data, the results indicate that Rab proteins and PI4P collaborate in the association of secretory compartments with Myo2p. Thus, we show that a coincidence detection mechanism coordinates inputs from PI4P and the appropriate Rab for secretory compartment transport.     

Tuberin is a component of lipid rafts and mediates caveolin-1 localization: role of TSC2 in post-Golgi transport

Mutations of the TSC2 gene lead to the development of hamartomas in tuberous sclerosis complex. Their pathology exhibits features indicative of defects in cell growth, proliferation, differentiation, and migration. We have previously shown that tuberin, the TSC2 protein, resides in multiple subcellular compartments and as such may serve multiple functions. To further characterize the microsomal pool of tuberin, we found that it cofractionated with caveolin-1 in a low-density, Triton X-100-resistant fraction (i.e., lipid rafts) and regulated its localization. In cells lacking tuberin, most of the endogenous caveolin-1 was displaced from the plasma membrane to a Brefeldin-A-sensitive, post-Golgi compartment distinct from the endosome and lysosome. Correspondingly, there was a paucity of caveolae at the plasma membrane of Tsc2-/- cells. Reintroduction of TSC2, but not a disease-causing mutant, reversed the caveolin-1 localization to the membrane. Exogenously expressed caveolin-1-GFP and vesicular stomatitis virus G protein, VSVG-GFP in the Tsc2-/- cells failed to be transported to the plasma membrane and were retained in distinct post-Golgi vesicles. Our data suggest a role of tuberin in regulating post-Golgi transport without apparent effects on protein sorting. The presence of mislocalized proteins in Tsc2-/- cells may contribute to the abnormal signaling and cellular phenotype of tuberous sclerosis.     

A role for caveolin-1 in post-injury reactive neuronal plasticity

Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin-1 is a cholesterol-binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up-regulation of caveolin-1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin-1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin-1-overexpressing cells revealed increases in synaptophysin and GAP-43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin-1-overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin-1-overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin-1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin-1 plays an active role in the regulation of injury-induced synaptic and terminal remodeling in the adult CNS.     

Depletion of Suds3 reveals an essential role in early lineage specification

Preimplantation development culminates with the emergence of three distinct populations: the inner cell mass, primitive endoderm and trophectoderm. Here, we define the mechanisms underlying the requirement of Suds3 in pre/peri-implantation development. Suds3 knockdown blastocysts exhibit a failure of both trophectoderm proliferation as well as a conspicuous lack of primitive endoderm. Expression of essential lineage factors Nanog, Sox2, Cdx2, Eomes, Elf5 and Sox17 are severely reduced in the absence of Suds3. Importantly, we document deficient FGF4/ERK signaling and show that exogenous FGF4 rescues primitive endoderm formation and trophectoderm proliferation in Suds3 knockdown blastocysts. We also show that Hdac1 knockdown reduces Sox2/FGF4/ERK signaling in blastocysts. Collectively, these data define a role for Suds3 in activation of FGF4/ERK signaling and determine an essential molecular role of Suds3/Sin3/HDAC complexes in lineage specification in vivo.     

Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite

Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.     

A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase.

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.     

Endothelial caveolin-1 plays a major role in the development of atherosclerosis

Clinical studies have established the important impact of atherosclerotic disease in Western societies. This disease is characterized by the accumulation of lipids and the migration of various cell types in the sub-endothelial space of blood vessels. As demonstrated by many studies, endothelial cells play an essential role in the development of this disease. The endothelium acts as a gatekeeper of blood vessel integrity and cardiovascular health status. For instance, the transfer of lipids via the transport of lipoproteins in the arterial intima is believed to be mediated by endothelial cells through a process termed transcytosis. In addition, lipoproteins that accumulate in the sub-endothelial space may also be modified, in a process that can direct the activation of endothelial cells. These steps are essential for the initiation of an atherosclerotic plaque and may be mediated, at least in part, by caveolae and their associated protein caveolin-1. In the present study, we evaluate the role of caveolin-1/caveolae in the regulation of these two steps in endothelial cells. Our data clearly demonstrate that caveolin-1 is involved in the regulation of lipoprotein transcytosis across endothelial cells and in the regulation of vascular inflammation.     

Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis.

BACKGROUND: During cytokinesis, the plasma membrane of the parent cell is resolved into the two plasma membranes of the daughter cells. Membrane fusion events mediated by the machinery that participates in intracellular vesicle trafficking might contribute to this process. Two classes of molecules that are required for membrane fusion are the t-SNAREs and the v-SNAREs. The t-SNAREs (syntaxins) comprise a multi-gene family that has been suggested to mediate, at least in part, selective membrane fusion events in the cell. RESULTS: We have analyzed the genome of Caenorhabditis elegans and identified eight syntaxin genes. RNA-mediated interference (RNAi) was used to produce embryos deficient in individual syntaxins and these embryos were phenotypically characterized. Embryos deficient in one syntaxin, Syn-4, became multinucleate because of defects in karyomere fusion and cytokinesis. Syn-4 localized both to ingressing cleavage furrows and to punctate structures surrounding nuclei as they reformed during interphase. CONCLUSIONS: Our analyses indicate that both cytokinesis and reformation of the nuclear envelope are dependent on SNARE-mediated membrane fusion.     

Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase

The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme. Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p. Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria. Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p. Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype. We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants. These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone.     

Structure-based functional analysis reveals a role for the SM protein Sly1p in retrograde transport to the endoplasmic reticulum

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.     

The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.     

Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis

delta-Aminolevulinic acid (ALA), the first committed precursor to the tetrapyrrole components of hemes and chlorophylls, is synthesized by two different routes in the photosynthetic phytoflagellate Euglena gracilis: directly from glutamate, mediated by a 5-carbon pathway, and via condensation of glycine and succinyl-CoA, catalyzed by the enzyme ALA synthase. The physiological roles of the two pathways were determined by administration of specifically 14C-labeled ALA precursors to cultures growing under different physiological conditions. Relative activities of the ALA synthase and 5-carbon pathways were monitored by incorporation of radioactivity from [2-14C] glycine and [1-14C]glutamate into highly purified protoheme, heme a and chlorophyll a derivatives. Wild type cells grown photoautotrophically or photoheterotrophically synthesized chlorophyll and incorporated radioactivity from [1-14C]glutamate into the tetrapyrrole nucleus of the pigment. [2-14C]Glycine was incorporated primarily into the nontetrapyrrole-derived portions of chlorophyll. In the same cultures both [2-14C]glycine and [1-14C]glutamate were efficiently incorporated into protoheme, while only [2-14C] glycine was incorporated into heme a. In dark-grown wild type or light-grown aplastidic cells, no chlorophyll was formed, and both protoheme and heme a were labeled exclusively from [2-14C]glycine. These results indicate: (a) ALA synthase and the 5-carbon pathway operate simultaneously in growing green cells; (b) the 5-carbon pathway provides ALA for chloroplast protoheme and chlorophyll, and is associated with chloroplast development; (c) ALA synthase provides ALA only for nonplastid heme biosynthesis; and (d) the two ALA pathways are separately compartmentalized along with complete sets of enzymes for subsequent tetrapyrrole synthesis from each ALA pool. The protoheme that was synthesized from [1-14C] glutamate had a higher specific radioactivity than chlorophyll synthesized from the same precursor. This result together with calculated specific radioactivities of the products synthesized during the incubation period, suggest that both protoheme and heme a undergo metabolic turnover.     

The identification of CVP1 reveals a role for sterols in vascular patterning

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.