Biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites

Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle's cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis

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

Prohibitin, an antiproliferative protein, is localized to mitochondria

AbstractProhibitin is a ubiquitously expressed protein with antiproliferative properties. When rat prohibitin tagged with a carboxy-terminal c-Myc epitope was expressed in baby hamster kidney cells the protein was targeted to mitochondria. In immunofluorescence microscopy prohibitin colocalized with a mitochondrial marker E3. Immunoelectron microscopy revealed that prohibitin was associated with the periphery of mitochondria. The amino-terminus of prohibitin shares characteristics of the known mitochondrial import signals, and positioning of the tag at the N-terminus causes accumulation of the protein in the cytoplasm. These findings help to direct functional studies on prohibitin and suggest that a mitochondrial protein may act as a tumor suppressor

A Caveolin Dominant Negative Mutant Associates with Lipid Bodies and Induces Intracellular Cholesterol Imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts

Guidelines for the use of diagnostic imaging in musculoskeletal pain conditions affecting the lower back, knee and shoulder: A scoping review

Background Musculoskeletal (MSK) pain is one of the most common reasons for primary care consultation, particularly pain in the lower back (LBP), knee and shoulder. The use of diagnostic imaging for MSK pain is increasing, but it is unclear whether this increase is justified on the basis of clinical practice guideline (CPG) recommendations. Aim To identify and map the content of CPGs that informs the use of diagnostic imaging in those with nontraumatic LBP, knee and shoulder pain in primary and intermediate care in the UK. Design and Setting A scoping review of CPGs. Methods This scoping review was conducted and is reported in accordance with PRISMA guidance. A broad search strategy included electronic searches of MEDLINE, CINAHL, PsychINFO and SPORTDiscus from 2009 to 17 April 2019. This was conducted alongside a search of guideline repositories and was combined with a snowball search of Google, relevant professional bodies and use of social media. Results 31 relevant CPGs were included. Routine use of diagnostic imaging for those with nontraumatic LBP, knee or shoulder pain is generally discouraged in primary care or intermediate care. Diagnostic imaging should be reserved for when specific or serious pathology is suspected or where the person is not responding to initial nonsurgical management and the imaging result is expected to change clinical management decisions. Conclusion Diagnostic imaging should not be routinely requested in primary or intermediate care for nontraumatic LBP, knee or shoulder pain. CPGs do not justify the increasing imaging rates in the UK for MSK pain

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins

Internalization and recycling of activated thrombin receptors

Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, thrombin receptors undergo homologous desensitization, temporarily losing their ability to respond to both agonists. We have examined the role of receptor internalization and recycling in this process using receptor-directed antibodies as probes. The results show within 1 min of activation >85% of the approximately 200,000 thrombin receptors on megakaryoblastic human erythroleukemia (HEL) and CHRF-288 cells are sequestered into endosomes via coated pits, after which the majority are transferred to lysosomes. This process does not require proteolysis of the receptor and occurs with sufficient speed to play a major role in the regulation of thrombin receptor function. Although most of the internalized receptors are ultimately degraded, approximately 25% return to the cell surface. These recycled receptors are in a state in which they can respond to SFLLRN but not thrombin; nor do they self-activate despite the apparent continued presence of the tethered ligand. In contrast to other G protein-coupled receptors, which are internalized and then recycled in an activatable state, recovery of the thrombin response occurs only after the expression on the cell surface of adequate numbers of newly synthesized receptors

Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes

Augmented Reality for Deaf Students: Can Mobile Devices Make it Possible?

Abstract. Digital and real world events can be combined to create powerful learning opportunities for students, but time, tools, and expertise have been traditional barriers to teacher-created enhancements. This paper provides a rationale for using emerging, teacher-friendly tools, to merge real space and virtual space through video and 2D barcodes. The results of three pilot studies combine to illustrate the potential for using these tools. Results indicate that cell phones have the potential to facilitate augmented reality experiences for deaf students and adults