A dynamic partitioning mechanism polarizes membrane protein distribution.

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose "dynamic partitioning" as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

Styrene maleic acid recovers proteins from mammalian cells and tissues while avoiding significant cell death.

Detection of protein biomarkers is an important tool for medical diagnostics, typically exploiting concentration of particular biomarkers or biomarker release from tissues. We sought to establish whether proteins not normally released by living cells can be extracted without harming cells, with a view to extending this into biomarker harvest for medical diagnosis and other applications. Styrene maleic acid (SMA) is a polymer that extracts nanodiscs of biological membranes (containing membrane proteins) from cells. Hitherto it has been used to harvest SMA-lipid-membrane protein particles (SMALP) for biochemical study, by destroying the living cellular specimen. In this study, we applied SMA at low concentration to human primary cardiovascular cells and rat vascular tissue, to 'biopsy' cell proteins while avoiding significant reductions in cell viability. SMA at 6.25 parts per million harvested proteins from cells and tissues without causing significant release of cytosolic dye (calcein) or reduction in cell viability at 24 and 72 hours post-SMA (MTT assay). A wide range of proteins were recovered (20-200 kDa) and a number identified by mass spectrometry: this confirmed protein recovery from plasma membrane, intracellular membranes and cell cytosol without associated cell death. These data demonstrate the feasibility of non-lethally sampling proteins from cells, greatly extending our sampling capability, which could yield new physiological and/or pathological biomarkers.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

Evaluación de lípidos y lipoproteínas en escolares residentes de gran altitud / Evaluation of lipids and lipoproteins in school children high altitude residents

PREGUNTA DE INVESTIGACIÓN: ¿Cuáles serán los valores de lípidos y lipoproteínas en niños y niñas en edad escolar, de zona periférica de La Paz, Bolivia, residentes de altitud, en la gestión 2011? OBJETIVO: establecer los valores de lípidos y lipoproteínas en niños y niñas en edad escolar de zona periférica de La Paz, Bolivia, residentes de altitud, en la gestión 2011. MATERIAL Y MÉTODOS: estudio descriptivo transversal, en 84 escolares de 6 a 13 años de edad. Realizado en zona periurbana y otra no periurbana, ciudad de La Paz, a 3700 metros de altitud. Se realizaron, examen clínico pediátrico; peso, talla, índice de masa corporal (IMC) (kg/talla2), pliegues tricipital, subescapular, suprailíaco; perímetro de cintura, y dosificación de triglicéridos, colesterol total, lipoproteína de alta densidad, HDL-c y de baja densidad, LDL-c. RESULTADOS: la obesidad era de 8% (IMC-Z > 2), la circunferencia de cintura, estaba incrementada en 24 escolares (28%), a predominio de varones. Los varones tenían valores promedio más elevados de colesterol total, LDL-c y HDL-c que las mujeres, diferencia estadísticamente significativa. Acorde a referencias de poblaciones de nivel del mar, se encontró triglicéridos, en < 10 años, elevados en 13 escolares (31 %). HDL-c baja en 28 escolares (33%). Nivel socio económico (NSE) bajo, en < 10 años, triglicéridos elevados, 11 escolares (34%); HDL-c baja, 23 escolares (38 %). NSE medio, HDL-c baja, 5 escolares (21 %). En mujeres, en < 10 años, triglicéridos elevados en 6 escolares (24%). HDL-c baja en 19 escolares (40%). En varones, en < 10 años, triglicéridos elevados en 7 escolares (41%), y HDL-c baja en 9 escolares (24%). CONCLUSIONES: escolares de gran altitud presentaron valores elevados de triglicéridos, a predominio de los menores de 10 años, de un NSE bajo; las lipoproteínas HDL-c estaban disminuidas en estos mismos grupos. La prevención primaria de los factores de riesgo, debe ser uno de los principales propósitos, de alta prioridad, de las estrategias de salud escolar en nuestro contexto de altitud.

RESEARCH QUESTION: which are the values of lipids and lipoproteins values of school children high altitude residents in peripheral areas of La Paz city, Bolivia, 2011? OBJECTIVE: To determine lipids and lipoproteins values of schoolchildren high altitude residents in peripheral areas of La Paz city, Bolivia, 2011 METHODS: a descriptive, transversal study was conducted in urban and periurban areas of La Paz city at 3700 meters above sea level. The study included 84 schoolchildren between 6 to 13 years old. A pediatric clinic examination and anthropometric measurements such as weight, height, skin folds and circumferences were performed. Cholesterol, triglycerides, high and low density lipoproteins (HDL-c and LDL-c) were determinate by conventional methods. RESULTS: We found obesity in 8% of school children defined by BMI-age Z score > 2 SD, waist circumference was increased in 24 subjects (28%), with male predomination. Total cholesterol, HDL-c and LDL-c were higher in males than females, difference statistic significant (p=0.05). According to references values at sea level populations, the triglycerides were higher in 13 subjects younger than 10 years (31%), HDL-c was low in 28 subjects (33%). By socioeconomic level we found in the low group high values of triglycerides in 11 subjects younger than 10 years and HDL-c low in 23 subjects (38%), in the medium group we found HDL-c low in 5 subjects (21%). By sex in females we found triglycerides high in 6 subjects younger than 10 years and HDL-c low in 19 subjects (40%), in males younger than 10 years we found triglycerides high in 7 subjects (41%) and HDL-c low in 9 subjects (24%). CONCLUSIONS: school children living at high altitude present high values of triglycerides, in subjects under 10 years old with a low socioeconomic level and HDLc were low in this same groups. The primary prevention of risk factors against cardiovascular diseases must to be a priority in health scholar strategies in our context.

Involvement of Src in the membrane skeletal complex, MPP6-4.1G, in Schmidt-Lanterman incisures of mouse myelinated nerve fibers in PNS.

Schmidt-Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). In this study, we report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite to those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the membrane protein palmitoylated 6 (MPP6) in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate that the Src-MPP6-4.1G protein complex in SLIs has a role in signal transduction in the PNS.

Essential function of protein 4.1G in targeting of membrane protein palmitoylated 6 into Schmidt-Lanterman incisures in myelinated nerves.

Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures.

A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.

Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.

Identification and characterization of oxylipid-protein and peptide conjugates by mass spectrometry.

The modification of proteins by reactive products of lipid peroxidation is associated with a large number of diseases and biological aging; thus, methods that enable the characterization of oxylipid-protein and/or peptide conjugates are highly in demand. This unit outlines a chemical labeling approach to identifying and characterizing proteins modified by lipid peroxidation products. It also outlines two approaches for mass spectrometry-based identification and detailed characterization of oxylipid conjugates. The first combines chemical labeling of oxylipid-protein conjugates using an aldehyde-specific biotinylation reagent, electrophoretic separation, and mass spectrometry-based identification of the biotinylated proteins. In the second approach, protein extracts are treated with the aldehyde-specific reagent, proteolyzed using trypsin, and the biotinylated peptides are enriched using immobilized monomeric avidin. The enriched peptide fractions are submitted to tandem mass spectrometry for determining the peptide sequence information, site of the modification, and chemical nature of the oxylipid.