Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain

Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.     

Physiological Mechanisms Underlying Fruit Sunburn

Sunburn is a physiological disorder that can be observed in fruits of several crops growing in areas with warm climates, as a result of photodamage due to an excess of heat and/or light irradiance (visible and ultraviolet light). The main cause is thought to be an increase in reactive oxygen species production which causes oxidative damage due to the incapacity of the fruit to recover from stress. This can result in a characteristic morphological and structural phenotype unacceptable to consumers, leading to severe losses in productivity for farmers. Fruits have a great array of mechanisms to mitigate or reduce reactive oxygen species production and the inactivation of photosynthetic apparatus, such as enhanced xanthophyll cycle-dependent energy dissipation, accumulation of photoprotective pigments and heat-shock proteins, and the biosynthesis of antioxidants, among others. Nevertheless, these mechanisms become inefficient when the stress factors altering the fruit surface exceed a certain threshold (of both duration and intensity). Although this disorder has been studied in detail and previous efforts have provided significant advances in understanding the underlying mechanisms causing sunburn in a number of fruits, further research is still needed. This will undoubtedly provide new approaches and tools for improving current mitigation strategies.     

Hormonal Profiling Reveals a Hormonal Cross-Talk During Fruit Decay in Sweet Cherries

Journal of Plant Growth Regulation - Although fruit decay in sweet cherries has been studied in some detail, information is still scarce about the possible role of phytohormones during postharvest....     

Modulation of A-NK cell rigidity: In vitro characterization and in vivo implications for cell delivery

The delivery of cells to specific regions of the vasculature is a critical step in many therapeutic strategies. These include the packaging of DNA or RNA in cell "vehicles" for delivery to tissues, the reconstitution of differentiated cells to an organ using embryonic stem cells, and the enhancement of the immune response using effector lymphocytes. In most cases, these cells must be injected systemically. Unfortunately, ex vivo manipulation or activation can affect cell visco-elastic properties, making it difficult for the injected cells to traverse capillary beds. Compounding the problem is the fact that common agents used in the laboratory for increasing cell deformability generally have adverse side effects on the therapeutic potential of the cells. Using micropipet aspiration techniques, cytotoxicity assays and in vivo trafficking studies we show that: (1) the rigidity of injected effector cells directly affects resistance to passage through tissue; (2) modulation of cytoskeletal organization can be used to decrease cell rigidity, but can also compromise therapeutic efficacy; and (3) thioglycollate, an agent which does not influence effector lymphocyte cytotoxic activity, reduces cell rigidity and entrapment in the lungs.     

The morphology and histology of the cirripede pemis

The general morphology and detailed histology of the penis of two common boreo-arctic cirripedes, Balanus balanoides (L.) and B. balanus (L.) have been investigated. The penis is a highly extensible, annulated organ beset with four rows of sensory setae. The paired vesiculae seminales unite within the pedicel of the penis to give the single ductus. Distally, the exoskeleton is invaginated into this ductus. Circular muscles are present in the vesiculae seminales but do not continue into the penis. The histology of the ductus epithelium indicates a secretory nature as does that of a specialized group of cells, termed the ‘cushion’, towards the distal end: this group of cells is surrounded by circular muscle bands. Longitudinal muscles extend virtually the whole length of the penis; they give off fibres which are inserted at the junctions of the annulations. The muscles of the pedicel are described. Paired nerves in the pedicel give rise to four in the penis. The sensory innervation of the setae is described. The possible functional relations of the structure to the activities of the penis at copulation and during the emission of semen is discussed.     

Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.     

Proteins, recognition networks and developing interfaces for macromolecular biosensing

Genomics and proteomics discovery is leading to the identification of all proteins and to the opportunity, and challenge, to reveal the protein recognition networks that drive virtually all biological processes. Over the past decade, biosensors have emerged as a key technology for detection and analysis of biomolecular interactions. An important limitation in developing such biosensors is that the focus has been mainly on sensor platforms, the transducing hardware that converts interaction signals into recorded data, without adequately considering the role of molecular interfaces, the elements of sensors that interact with analytes to produce signals. We have investigated this alternative focus by identifying and, where necessary, designing molecular interfaces that will more effectively drive new biosensor development and utilization in biomedical and biotechnological investigations. Here we describe our recent studies of coiled coil and lipid bilayer interfaces and the potential to use these to expand sensing technologies for multiplexed target detection and analysis in increasingly biologically relevant membrane like environments.