Biology subjects

Cullen, P. J.

Publications and source records attributed to Cullen, P. J..

5 recordsLinked to original sources

bioRxiv2019

Acute perturbation of retromer and ESCPE-1 leads to functionally distinct and temporally resolved defects in endosomal cargo sorting.

The human retromer is a stable heterotrimer of VPS35, VPS29 and VPS26 whose principal role is to orchestrate the endosomal retrieval of hundreds of internalised cargo and promote their recycling to the cell surface; a prototypical cargo being the glucose transporter GLUT1. Retromers role in a distinct endosomal retrieval pathway, the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network (TGN), remains controversial. Here we have developed and applied knocksideways to acutely inactivate retromer and by visualising the sorting of endogenous GLUT1 and CI-MPR provide insight into the temporal dynamics of endosomal cargo sorting in HeLa and H4 human neuroglioma cells. While retromer knocksideways led to the development of time-resolved defects in cell surface sorting of GLUT1 we failed to observe defects in the sorting of the CI-MPR. In contrast knocksideways of ESCPE-1, a key regulator of retrograde CI-MPR sorting, resulted in a time-resolved defect in CI-MPR sorting. Together these data provide independent evidence consistent with a comparatively limited role for retromer in ESCPE-1 dependent CI-MPR retrograde sorting in HeLa and H4 human neuroglioma cells.

cell biology↗

bioRxiv2019

Low dose Cold Atmospheric Plasma induces membrane oxidation, stimulates endocytosis and enhances uptake of nanomaterials in Glioblastoma multiforme cells

Cold atmospheric plasma (CAP) has demonstrated synergistic cytotoxic effects with nanoparticles, especially promoting the uptake and accumulation of nanoparticles inside cells. However, the mechanisms driving the effects need to be explored. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP (30 s, 75 kV). CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidated lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrin-dependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. Our data demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake which will be useful for developing more efficient deliveries of nanoparticles and pharmaceuticals into cancer cells for tumour therapy and diagnosis. This mechanism of RONS-induced endocytosis will also be of relevance to other cancer therapies that induce an increase in extracellular RONS.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/805192v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (43K):\norg.highwire.dtl.DTLVardef@1487fd4org.highwire.dtl.DTLVardef@c05eeforg.highwire.dtl.DTLVardef@198c75aorg.highwire.dtl.DTLVardef@1481096_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

Regulation of Bud Emergence by a MAPK Pathway

All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in yeast, where the Rho GTPase Cdc42p regulates symmetry breaking at bud sites and the establishment of polarity by interacting with effector proteins. The prevailing view of bud emergence does not account for regulation by extrinsic cues or signal transduction pathways. Here, we show that the MAPK pathway that controls filamentous growth (fMAPK pathway), which also requires Cdc42p and the effector p21 activated kinase (PAK) Ste20p, regulates bud emergence under nutrient-limiting conditions that favor filamentous/invasive growth. The fMAPK pathway regulated the expression of polarity targets that included the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and highest at a period in the cell cycle that coincided with bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced growth at multiple sites that resulted from multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

cell biology↗

bioRxiv2019

Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

Rho GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous ({sum}1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared to the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. In the fMAPK pathway, Bem1p recruited Ste20p to the plasma membrane, cycled between an open and closed conformation, and interacted with the GEF for Cdc42, Cdc24p. Bem1p also regulated effector pathways in different ways, behaving as a multi-functional adaptor in some pathways and an inert scaffold in others. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.\n\nHIGHLIGHTSO_LIComparing Cdc42p-dependent MAPK pathways showed that the fMAPK pathway had slow activation kinetics compared to the mating and HOG pathways.\nC_LIO_LIA collection of cdc42 alleles was tested for MAPK pathway functions.[§] Cdc42pE100A, previously characterized as being specifically defective for fMAPK signaling, showed reduced interaction with the fMAPK pathway adaptor Bem4p.\n[§]Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK signaling.\n\nC_LIO_LIThe polarity adaptor Bem1p regulated the fMAPK pathway.[§] Bem1p regulated the fMAPK pathway by recruiting Ste20p to the plasma membrane, cycling between an open and closed conformation, and interacting with the Cdc42p GEF, Cdc24p.\n\nC_LIO_LIDifferent domains of Bem1p had different roles in regulating effector pathways.[§] Bem1p may function as a multi-functional adaptor in some pathways and an inert scaffold in others.\n\nC_LIO_LIBem4p and Bem1p regulated the fMAPK pathway in an ordered sequence.[§] The data support a model where Bem4p recruits Cdc24p to GDP-Cdc42p, and Bem1p directs GTP-Cdc42p to Ste20p at the plasma membrane.\n[§]The bud-site GTPase Rsr1p regulates Cdc24p in the fMAPK pathway but does not initiate signaling.\n\nC_LI

genetics↗

bioRxiv2019

Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton

Transmembrane mucin-type glycoproteins can regulate signal transduction pathways. In yeast, signaling mucins regulate mitogen-activated protein kinase (MAPK) pathways that induce cell differentiation to filamentous growth (fMAPK pathway) and the response to osmotic stress (HOG pathway). To explore regulatory aspects of signaling mucin function, protein microarrays were used to identify proteins that interact with the cytoplasmic domain of the mucin-like glycoprotein, Msb2p. Eighteen proteins were identified that comprised functional categories of metabolism, actin filament capping and depolymerization, aerobic and anaerobic growth, chromatin organization and bud growth, sporulation, ribosome biogenesis, protein modification by iron-sulfur clusters, RNA catabolism, and DNA replication and DNA repair. A subunit of actin capping protein, Cap2p, interacted with the cytoplasmic domain of Msb2p. Cells lacking Cap2p showed altered localization of Msb2p and increased shedding of Msb2ps N-terminal glycosylated domain. Consistent with its role in regulating the actin cytoskeleton, Cap2p, and another Msb2p-interacting protein, Aip1p, were required for the enhanced cell polarization during filamentous growth. Our study identifies proteins that connect a signalling mucin to diverse cellular processes and may provide insight into new aspects of mucin function.

biochemistry↗