Protein Kinase D-Mediated Anterograde Membrane Trafficking Is Required for Fibroblast Motility

AbstractBackground: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.Results: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.Conclusions: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration

Alanine-scanning mutagenesis of Aspergillus γ-tubulin yields diverse and novel phenotypes

This is the publisher's version, also available electronically from "www.molbiolcell.org".We have created 41 clustered charged-to-alanine scanning mutations of the mipA, γ-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of γ-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that γ-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of γ-tubulin and these data give a good initial indication of the functionally important regions of the molecule

The use of beta-D-glucanase as a substitute for Novozyme 234 in immunofluorescence and protoplasting

Novozym 234 has been used for many years to prepare protoplasts of Aspergillus nidulans and other fungi for transformation. It has also been very useful in immunofluorescence studies for partially digesting walls of fixed hyphae or germlings to allow antibodies to penetrate into the cytoplasm. In recent years, the availability of Novozym 234 has become problematic, and we have searched for combinations of available enzymes that are suitable for protoplasting and immunofluorescence studies in A. nidulans

Detection and Characterization of Circulating Tumour Cells in Multiple Myeloma

Multiple myeloma (MM) remains an incurable disease despite recent therapeutic improvements. The ability to detect and characterize MM circulating tumour cells (CTCs) in peripheral blood provides an alternative to replace or augment invasive bone marrow (BM) biopsies with a simple blood draw, providing real-time, clinically relevant information leading to improved disease management and therapy selection. Here we have developed and qualified an enrichment-free, cell-based immunofluorescence MM CTC assay that utilizes an automated digital pathology algorithm to distinguish MM CTCs from white blood cells (WBCs) on the basis of CD138 and CD45 expression levels, as well as a number of morphological parameters. These MM CTCs were further characterized for expression of phospho-ribosomal protein S6 (pS6) as a readout for PI3K/AKT pathway activation. Clinical feasibility of the assay was established by testing blood samples from a small cohort of patients, where we detected populations of both CD138pos and CD138neg MM CTCs. In this study, we developed an immunofluorescent cell-based assay to detect and characterize CTCs in MM

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications

PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting