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Sculpting membranes into dynamic, curved shapes is central to intracellular cargo trafficking and other cellular functions. However, generation of membrane curvature during trafficking involves lipids and membrane-associated proteins; current mechanisms focus on creating rigid cages, curved scaffolds, or membrane surface area changes by proteins. This dissertation provides an alternative mechanistic example for the control of membrane deformations, involving modulation of membrane material properties. Sar1, a GTPase of the COPII family, regulates vesicle trafficking from the endoplasmic reticulum. We find that Sar1p lowers the rigidity of the lipid bilayer membrane to which it binds. We examine the behavior of Saccharomyces cerevisiae Sar1 (Sar1p) and Homo sapiens paralogs of Sar1 (Sar1A and Sar1B). Like Sar1p, human Sar1s lower membrane rigidity. Unlike Sar1p, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which human Sar1 clustering governs membrane mechanical properties. Membrane rigidity measurements remain rare, however, and show a large variance, a situation that can be addressed by improving techniques and comparing disparate techniques applied to the same systems. I introduce applying selective plane illumination microscopy (SPIM) to image thermal fluctuations of giant vesicles. SPIM's optical sectioning enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, we show rigidity values from giant unilamellar vesicle fluctuations in close agreement with those derived from our independent assay based on membrane tether pulling. We also show that a model of homogeneous quasi-spherical vesicles poorly fits fluctuation spectra of vesicles bound by Sar1A at high concentrations, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties. I conclude by discussing our current work on amphipathic alpha helices, their ability to reduce membrane rigidity, and our hopes to create artificial helical structures capable of mimicking trafficking systems. Supplemental videos represent membrane disintegration with Sar1p and fluctuations of membrane only and Sar1p incubated vesicles. This dissertation contains previously published co-authored material.

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The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...

<p>Vesicle trafficking is an important process critical for secretory and endocytic purposes, but it is also crucial for cell homeostasis, <i>e.g.,</i> for maintenance of organelle identity and recycling of membrane components.</p><p>The endomembrane-located adaptor protein Epsin R (Epsin-Related protein) is believed to be important for recycling of SNARES like Vti1b from endosomes to the trans Golgi network (TGN), although its involvement in TGN to endosome transport has been also proposed. Further highlighting its impact in cellular and organismal physiology, certain <i>EPSIN R</i> SNPs have been linked to schizophrenia and Epsin R deficiencies correlate with other pathological conditions related to epidermis homeostasis such as psoriasis and eczema.</p><p>Epsin R belongs to the conserved Epsin family of adaptors and as such it presents a characteristic Epsin N-Terminal Homology (ENTH) domain and a largely unstructured C-terminus. The latter contains binding motifs for important elements of the vesicle trafficking machinery.</p><p>Here we identified a C-terminal region of Epsin R with prion-like characteristics (Prion Forming Region or PFR). We found that GFP-Epsin R is localized in intracellular punctate structures colocalizing with different intracellular markers; however, in contrast to other epsin family members, Epsin R displayed puncta of different size and with different protein content with a substantial contribution of large/bright particles. Importantly, the C-terminal Epsin R’s PFR was required for Epsin R localization and for the formation of large and bright puncta. Further, these structures displayed characteristics shared with other prion-like proteins. Our results therefore suggest that Epsin R possesses PFR-dependent prion properties that play an important role in this adaptor’s localization and function.</p><p>We propose a model in which prion-like proteins like Epsin R can rapidly and stably self-assemble at vesicle budding sites. These proteins would accelerate the formation of vesicle trafficking machinery and the recruitment of cargo. We also speculate that oligomerizing, self-templating reactions would occur under strict control of several cellular factors such as chaperones and post-translational modifications (<i>e.g.,</i> phosphorylation, ubiquitination, etc.) to assure quick and <i>reversible</i> association of prion-like proteins.</p>

Compatible interactions between male gametophytes (pollen) and the female reproductive organ (pistil) are essential for fertilization in flowering plants. Recognition at a molecular level allows “compatible” pollen grains to adhere/germinate on the stigma while pollen grains from unrelated plant species are largely ignored. The exocyst is a large eight subunit complex that is primarily involved in polarized secretion or regulated exocytosis in eukaryotic cells where it functions to tether vesicles to the plasma membrane. Recent research has implicated one of the Exo70 family members, Exo70A1, in compatible pollen-pistil interactions in Arabidopsis and Brassica. The loss of Exo70A1 in Arabidopsis Col-0 stigmas leads to the rejection of compatible pollen producing a “female sterile” phenotype. Through my research I have demonstrated that, driven by a stigma-specific promoter, an RFP:Exo70A1 fusion protein rescues this defect in exo70A1-1 mutant and Exo70A1 is found to be localized to the plasma membrane at flower opening.

Compatible interactions between male gametophytes (pollen) and the female reproductive organ (pistil) are essential for fertilization in flowering plants. Recognition at a molecular level allows “compatible” pollen grains to adhere/germinate on the stigma while pollen grains from unrelated plant species are largely ignored. The exocyst is a large eight subunit complex that is primarily involved in polarized secretion or regulated exocytosis in eukaryotic cells where it functions to tether vesicles to the plasma membrane. Recent research has implicated one of the Exo70 family members, Exo70A1, in compatible pollen-pistil interactions in Arabidopsis and Brassica. The loss of Exo70A1 in Arabidopsis Col-0 stigmas leads to the rejection of compatible pollen producing a “female sterile” phenotype. Through my research I have demonstrated that, driven by a stigma-specific promoter, an RFP:Exo70A1 fusion protein rescues this defect in exo70A1-1 mutant and Exo70A1 is found to be localized to the plasma membrane at flower opening.

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

Vor zehn Jahren wurde der (Pro)Renin-Rezeptor [(P)RR] entdeckt und als neuer Bestandteil des Renin-Angiotensin-Systems beschrieben. Neuere Studien ergaben, dass der (P)RR mit der vakuolären H+-ATPase (V-ATPase) assoziiert sein kann, weshalb er auch V-ATPase associated protein 2 (ATP6ap2) genannt wird. In Osteoklasten befinden sich V-ATPase hauptsächlich an der zur Knochenoberfläche gerichteten Plasmamembran und transportieren Protonen in den extrazellulären Raum. Mäuse mit genetischer Deletion verschiedener V-ATPase-Untereinheiten charakterisiert durch einen Anstieg von Knochenmasse (Osteopetrose). In der vorliegenden Arbeit fanden wir heraus, dass (P)RR stark in reifen Osteoklasten in vitro und in vivo exprimiert wird. Mäuse mit genetischer Deletion des (P)RR in Osteoklasten wurden durch einen komplexen Knochen-Phänotyp mit reduzierter Knochendichte charakterisiert. (P)RR-defiziten Osteoklasten wiesen vermehrte Differenzierung und/oder Aktivität in vitro und in vivo auf. Wir postulieren deshalb, dass der (P)RR die in der Plasmamembran lokalisierten V-ATPase nicht direkt reguliert, sondern mit der physiologischen Aktivität der Osteoklasten durch andere Mechanismen interferiert. Macrophagen sind speziell auf die Immunabwehr ausgerichtete Fresszellen (Phagozyten). Phagozytose ist ein wesentlicher Zellprozess der die V-ATPase in Lysosomen braucht um die eingeschlossenen Pathogen zu zerstören. Wir generierten transgene Ratten mit konditionellen knockdown von (P)RR unter Nutzung eines Doxyzyclin-induzierten shRNA-Expressionssystems. Eine effiziente (P)RR-Depletion in Makrophagen wurde durch Behandlung mit Doxyzyclin in vivo im Trinkwasser und in vitro im Kulturmedium erreicht. Die vorliegende Arbeit zeigt, dass die Verschiebung des vesikulären pHs erst ziemlich spät nach (P)RR-Depletion auftritt. Wir fanden heraus, dass (P)RR-Depletion weder Phagozytose noch Endozytose beeinträchtigte, sondern für das Recycling des Transferrin-Rezeptors zur Plasmamembran wichtig ist. / A decade ago, the (pro)renin receptor [(P)RR] was discovered and depicted as a new component of the renin-angiotensin system. However, recent studies have put in evidence that the (P)RR associate with and regulate the vacuolar H+-ATPase (V-ATPase), hence its other name vacuolar H+-ATPase associated protein 2 (ATP6ap2). In osteoclasts, V-ATPases are mainly located at the plasma membrane facing the bone surface and extrude protons into the extracellular space. Mice with genetic deletion of various V-ATPase subunits are characterized by an increase of bone mass (osteopetrosis). In this work, we found that the (P)RR is highly expressed in mature osteoclasts in vitro and in vivo. Mice with genetic deletion of the (P)RR in osteoclasts developed a complex bone phenotype characterized by a reduced bone density. Osteoclasts lacking (P)RR displayed increased differentiation and/or activity in vitro and in vivo. We therefore suggest that the (P)RR does not directly regulate V-ATPases located at the plasma membrane but rather interferes with osteoclast physiology through other mechanisms. Macrophages are professionalized phagocytes crucial for immune response. Phagocytosis is an essential cellular process, which requires lysosomal V-ATPases for degradation of engulfed pathogens. We generated transgenic rats with a conditional depletion of the (P)RR with the use of a doxycycline-induced shRNA expression system. Efficient (P)RR depletion in macrophages was accomplished by doxycycline treatment in vivo in drinking water and in vitro in culture medium. In this work, we found that the impairment of vesicular pH occurs lately after (P)RR deletion. Also, we found that (P)RR deletion did not impair neither phagocytosis nor endocytosis but rather perturbed the recycling of the transferrin receptor to the plasma membrane.