Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis.

BACKGROUND: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. AIM: To characterize the effects of Prom-2 expression on PM microdomain organization. METHODS: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. RESULTS: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. CONCLUSIONS: Prom2 protrusions primarily localize to lipid rafts and recruit cholesterol into protrusions and away from caveolae, leading to increased phosphorylation of caveolin-1, which inhibits Cdc42-dependent endocytosis. This study provides a new insight for the role for prominins in the regulation of PM lipid organization.

Annexin A2 Mediates Dysferlin Accumulation and Muscle Cell Membrane Repair.

Muscle cell plasma membrane is frequently damaged by mechanical activity, and its repair requires the membrane protein dysferlin. We previously identified that, similar to dysferlin deficit, lack of annexin A2 (AnxA2) also impairs repair of skeletal myofibers. Here, we have studied the mechanism of AnxA2-mediated muscle cell membrane repair in cultured muscle cells. We find that injury-triggered increase in cytosolic calcium causes AnxA2 to bind dysferlin and accumulate on dysferlin-containing vesicles as well as with dysferlin at the site of membrane injury. AnxA2 accumulates on the injured plasma membrane in cholesterol-rich lipid microdomains and requires Src kinase activity and the presence of cholesterol. Lack of AnxA2 and its failure to translocate to the plasma membrane, both prevent calcium-triggered dysferlin translocation to the plasma membrane and compromise repair of the injured plasma membrane. Our studies identify that Anx2 senses calcium increase and injury-triggered change in plasma membrane cholesterol to facilitate dysferlin delivery and repair of the injured plasma membrane.

Antibody labeling of cholesterol/ceramide ordered domains in cell membranes.

A monoclonal antibody raised against mixed monolayers of 60:40 mol % cholesterol/C16-ceramide of known structure was used to label cholesterol/ceramide-rich domains in cell membranes. The antibody, Cer-Chol 405F specifically recognizes the mixed crystalline and homogeneous phase in monolayers, but it does not interact with either of the components separately. It interacts differently with mixed monolayers that contain ceramides of different acyl chain length. When used on cells, the antibody labeling is sensitive to changes in cholesterol and ceramide levels, as well as to over-expression of specific ceramides; this is in agreement with the results that were obtained on lipid monolayers. This represents a proof of concept of the applicability of a new approach to the structural characterization of lipid microdomains in cell membranes. The approach consists of raising antibodies that recognize specific structural organizations of lipids in artificial mixtures, characterizing the antibody/ordered domain complexes in vitro, and subsequently using them to detect the presence of the same (or similar) domains in cell membranes.

Mitochondrial redox signaling enables repair of injured skeletal muscle cells.

Strain and physical trauma to mechanically active cells, such as skeletal muscle myofibers, injures their plasma membranes, and mitochondrial function is required for their repair. We found that mitochondrial function was also needed for plasma membrane repair in myoblasts as well as nonmuscle cells, which depended on mitochondrial uptake of calcium through the mitochondrial calcium uniporter (MCU). Calcium uptake transiently increased the mitochondrial production of reactive oxygen species (ROS), which locally activated the guanosine triphosphatase (GTPase) RhoA, triggering F-actin accumulation at the site of injury and facilitating membrane repair. Blocking mitochondrial calcium uptake or ROS production prevented injury-triggered RhoA activation, actin polymerization, and plasma membrane repair. This repair mechanism was shared between myoblasts, nonmuscle cells, and mature skeletal myofibers. Quenching mitochondrial ROS in myofibers during eccentric exercise ex vivo caused increased damage to myofibers, resulting in a greater loss of muscle force. These results suggest a physiological role for mitochondria in plasma membrane repair in injured cells, a role that highlights a beneficial effect of ROS.

S100A11 is required for efficient plasma membrane repair and survival of invasive cancer cells.

Cell migration and invasion require increased plasma membrane dynamics and ability to navigate through dense stroma, thereby exposing plasma membrane to tremendous physical stress. Yet, it is largely unknown how metastatic cancer cells acquire an ability to cope with such stress. Here we show that S100A11, a calcium-binding protein upregulated in a variety of metastatic cancers, is essential for efficient plasma membrane repair and survival of highly motile cancer cells. Plasma membrane injury-induced entry of calcium into the cell triggers recruitment of S100A11 and Annexin A2 to the site of injury. We show that S100A11 in a complex with Annexin A2 helps reseal the plasma membrane by facilitating polymerization of cortical F-actin and excision of the damaged part of the plasma membrane. These data reveal plasma membrane repair in general and S100A11 and Annexin A2 in particular as new targets for the therapy of metastatic cancers.

Mechanism of Ca²âº-triggered ESCRT assembly and regulation of cell membrane repair.

In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

Ceramide transfer protein and cancer.

Sphingolipids are important structural components of membranes, and play an equally important role in basic cellular processes as second messengers. Recently, sphingolipids are receiving increasing attention in cancer research. Ceramide is the central molecule that regulates sphingolipid metabolism forming the basic structural backbone of sphingolipids and the precursor of all complex sphingolipids. It is been proposed to be an important regulator of tumor cell death following exposure to stress stimuli. The increase or decrease of ceramide levels leading to change in sensitivity of cancer cells to stress stimuli provides support for a central role of ceramide signaling in cell death. In this review, we have focused on ceramide transfer protein (CERT) as a major regulator of ceramide flux in the cell.

Two and three-dimensional pattern recognition of organized surfaces by specific antibodies.

Understanding molecular recognition of supramolecules for solid substrates is essential for designing chemical sensors and molecular devices. The rules of molecular recognition are well established at the level of single molecules. However, during the transition from molecular-scale devices to macroscopic devices, issues concerning control over recognition that are well-established at the molecular level become much more complex. Hopefully, the conceptual and practical considerations reported here will clarify some of these issues. The immune system uses antibodies to identify molecular surfaces through molecular recognition. Antibodies are thus appropriate tools to study the rules of macromolecule-surface interactions, and this was done using crystal surfaces as substrates. Crystals can be formed or introduced into organisms and should be thus treated by the organism as any other intruder, by eliciting antibodies specific to their surfaces. A structure-recognizing antibody is defined here as complementary to a certain ordered supramolecular organization. It can be considered as a mold bearing in its binding site memory of the organization against which it was elicited. On the surface of a crystal composed of relatively small organic molecules, an antibody binding site would encompass an array of 10-20 molecular moieties. The antibody binding site would not detect one molecule, but rather a two- or three-dimensional molecular arrangement on the surface, similar to a macromolecular surface. The complementarity between antibody binding site and surface is supported by stereoselective supramolecular interactions to the repetitive structural motifs that are exposed at the surface. A procedure was developed in order to isolate monoclonal antibodies that specifically recognize a certain crystalline surface. The procedure was applied in particular to crystals of cholesterol monohydrate, of 1,4-dinitrobenzene, and of the tripeptide (S)leucine-(S)leucine-(S)tyrosine (LLY). A series of antibodies were selected and studied, three of which provided reliable specific antibody-antigen structural models. The three docking models show an astounding geometrical and chemical match of the antibody binding sites on the respective crystal surfaces. We also showed that antibodies are intrinsically capable of recognition at the length scale necessary for detection of chirality. Once the structural parameters determining the antibody specificity to the target surfaces are characterized, the antibodies may be conceivably used as reporters of the existence and location of target domains with similar structure in biological milieus. In this context, we developed and characterized monoclonal antibodies specific to crystalline mixed monolayers of cholesterol and ceramide, fundamental building blocks of lipid microdomains in cellular membranes. When used on cells, one antibody indeed labels cell membrane domains composed of cholesterol and ceramide. The fundamental contribution of the approach developed here may be in the antibody ability to report on the structural organization of paracrystalline domains that cannot be determined by other means. Alternatively, structure-recognizing antibodies may be conceivably used to carry information or build connections to specific targets, which may offer interesting developments in medicine or electronics.

Structure of cholesterol/ceramide monolayer mixtures: implications to the molecular organization of lipid rafts.

The structure of monolayers of cholesterol/ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two components within a range of compositions of cholesterol/ceramide between 100:0 and 67:33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50:50 and 30:70; between 30:70 and 0:100 only the highly crystalline phase of ceramide was detected. The latter was determined and modeled. Immunolabeling was performed with an antibody specific to the cholesterol monohydrate crystalline arrangement. The antibody recognizes crystalline cholesterol monolayers, but does not interact with crystalline ceramide. Immunofluorescence and atomic force microscopy data show that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide. As ceramide incorporates the lipid backbone common to all sphingolipids, this arrangement may be relevant to the understanding of the molecular organization of lipid rafts.