Podcasts about pip2

Higher education institutions are facing a perfect storm of challenges that demand robust crisis management skills. From campus protests reminiscent of the 1960s to the lightning-fast spread of information (and misinformation) on social media, colleges and universities are navigating treacherous waters. How can they stay afloat – and even thrive – in this turbulent environment? Crisis Management in Higher Education: Strategies for Institutional Resilience Dr. Drumm McNaughton, CEO of The Change Leader higher education consulting firm, and guest Stephanie Craig, President of Kith crisis preparation, response, and recovery firm, discuss essential strategies for building crisis management and communication capabilities in higher education institutions. Key Discussion Points Higher Education's Unique Crisis Management Challenges: The complex stakeholder environment in higher education is reminiscent of the turbulent 1960s but with added dimensions of social media and 24-hour news cycles. The impact of poorly handled crises on various aspects of institutional operations, from student recruitment to donor confidence. Building Crisis Management Muscle: The PIP2 Approach: Plan: Develop comprehensive crisis strategies and response protocols. Invest: Allocating resources for crisis preparedness, including training and technology. Practice: Conducting regular crisis simulations to identify weaknesses and build muscle memory. Prioritize: Embedding crisis preparedness in institutional culture at all levels. The Clarity + Trust = Speed Formula: Clarity: Ensuring a clear understanding of institutional mission, values, and stakeholder priorities. Trust: Building trust within crisis management teams and with key stakeholders. Speed: Responding quickly and decisively to crises without sacrificing accuracy. Stakeholder Management in Higher Education Crises: Navigating the diverse and often conflicting interests of multiple stakeholder groups. Tailoring communication strategies to different stakeholder needs and preferences. The Role of Leadership and Second-Level Thinking: The importance of leadership visibility and engagement during crises. Employing "second-level thinking" to anticipate the ripple effects of decisions and communications. Integrating Risk Management and Crisis Communication: The collaboration between risk management and communication teams. Breaking down silos to ensure comprehensive crisis response strategies. Opportunities in Crisis: Catalysts for Institutional Innovation: Recognizing crises as potential drivers of positive change and innovation. Examples of institutional transformation sparked by crisis situations. Three Key Takeaways for Higher Education Presidents and Boards Invest in crisis management now, prioritizing preparation as a matter of urgency. Implement the PIP2 approach (Plan, Invest, Practice, Prioritize) to build crisis management muscle. Adopt the Clarity + Trust = Speed formula for effective crisis response.   Read the transcript on our website at: https://changinghighered.com/ building-higher-ed-crisis-management-and-communication-muscle/   #HigherEducation #HigherEdRiskManagement #CrisisManagement   About Our Podcast Guest Stephanie Craig is the President of Kith.co, where she provides strategic guidance and solutions for high-stakes issues and challenges faced by C-suite executives, high-profile organizations, and elected officials. She has built her reputation as a crisis expert by guiding some of the world's most prominent people and organizations through their most trying moments. Combining her deep experience in politics, media, and business, she mitigates crises, repairs reputations, and inoculates against future crises. Stephanie has counted former First Lady Rosalynn Carter, the mayor of the nation's 10th largest city, and some of the most notable global brands, as well as institutes of higher education, as clients. Stephanie is a prolific author on topics including crisis communications, reputational management, political communication, the intersection of law and communications, and entrepreneurship. If asked, she'll even talk about being born in the Arctic. About the Host Dr. Drumm McNaughton is the founder, CEO, and Principal Consultant at The Change Leader, Inc. A highly sought-after higher education consultant with 20+ years of experience, Dr. McNaughton works with leadership, management, and boards of both U.S. and international institutions. His expertise spans key areas, including accreditation, governance, strategic planning, presidential onboarding, mergers, acquisitions, and strategic alliances. Dr. McNaughton's approach combines a holistic methodology with a deep understanding of the contemporary and evolving challenges facing higher education institutions worldwide to ensure his clients succeed in their mission.   

References Int J Mol Sci. 2020 Jun; 21(11): 4098. Authentic Biochemistry lectures :ImmunoEpigenetics 56-58 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.19.533351v1?rss=1 Authors: Houngue, R., Sangare, L. O., Alayi, T. D., Dieng, A., Bitard-Feildel, T., Boulogne, C., Slomianny, C., Atindehou, C. M., Fanou, L. A., Hathout, Y., Saeij, J. P., Callebaut, I., Tomavo, S. Abstract: Apicomplexan parasites have specialized secretory organelles called rhoptries, micronemes, and dense granules that are essential for host infection. Here, we show that TgREMIND, a Toxoplasma gondii protein containing a membrane phospholipid interacting domain, is required for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain at the N-terminus, known to promote cell membrane bending, and a novel uncharacterized domain that we named REMIND for regulator of membrane interacting domain at the C-terminus. TgREMIND binds to PIP2 lipid species and both F-BAR and REMIND domains are necessary to ensure proper biological activities in vitro and in cellulo. Conditional depletion of TgREMIND results in the absence of dense granules and abnormal transparent rhoptries, leading to a severe inhibition of parasite motility, host invasion, and dissemination. Thus, our study demonstrates that TgREMIND is essential for the proper functioning of key secretory organelles required for successful infection by Toxoplasma. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.03.531032v1?rss=1 Authors: Thakore, P., Yamasaki, E., Ali, S., Sanchez Solano, A., Labelle-Dumais, C., Gao, X., Chaumeil, M. M., Gould, D. B., Earley, S. Abstract: Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.29.518389v1?rss=1 Authors: Moussa, H. Y. A., Park, Y. Abstract: Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10 to 100 uM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.21.517310v1?rss=1 Authors: Haase, D., Rasch, C., Keller, U., Elting, A., Wittmar, J., Janning, A., Kahms, M., Schuberth, C., Klingauf, J., Wedlich-Soldner, R. Abstract: Topography is a critical feature driving formation and dynamics of protein and lipid domains within biological membranes. The yeast plasma membrane (PM) has provided a powerful model system to study lateral domain formation, including characteristic BAR domain-induced PM furrows. Currently, it is not clear how the components involved in the establishment of these furrows cooperate to precisely regulate local PM topography. Here we report opposing functions for the Sur7 and Nce102 families of tetraspanner proteins in modulating membrane curvature and domain topography. Using STED nanoscopy and freeze-fracture EM we found that Sur7 tetraspanners form multimeric strands at the upper edges of PM furrows, which counteract the forces exerted by BAR domain proteins and prevent membrane tubulation. In contrast, Nce102 tetraspanners are located basal to the Sur7 proteins and promote BAR domain-induced curvature. The segregation of the two tetraspanner-based nanodomains is further supported by differential distribution of ergosterol to the upper edge of furrows and PIP2 lipids at the furrow base. These findings suggest a general role of tetraspanner proteins in sculpting local membrane domains. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512717v1?rss=1 Authors: Harvey, K. E., Tang, S., LaVigne, E. K., Pratt, E. P. S., Hockerman, G. H. Abstract: The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 M carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased by 65% in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates {beta}-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512101v1?rss=1 Authors: Padinjat, R., Saha, S., Krishnan, H. Abstract: Lithium (Li) is a widely used as a mood stabilizer in the clinical management of Bipolar Affective Disorder (BPAD). However, the molecular targets of Li in neural cells that underpin its therapeutic effect remain unresolved. Inositol monophosphatase (IMPA1), is an enzyme involved in the resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) following receptor-activated phospholipase C (PLC) signalling. In vitro, Li inhibits IMPA1, but the relevance of this inhibition within neural cells remains unknown. Here we report that in human cells, treatment with therapeutically relevant concentrations of Li reduces receptor activated calcium release from intracellular stores and also delays the resynthesis of PIP2 following receptor activated PLC signalling. Both these effects of Li are abrogated in cells where IMPA1 has been deleted. We also observed that in human forebrain cortical neurons, treatment with Li results in reduced neuronal excitability as well as reduced calcium signals following receptor activated PLC signalling. Following Li treatment of human forebrain cortical neurons, transcriptome analyses reveal downregulation of multiple components of the glutamate receptor signalling system. Glutamate is a key excitatory neurotransmitter in the human brain and thus our findings provide an insight into the mechanisms underlying the dampening of neuronal excitability following Li treatment. Collectively, our findings suggest that Li inhibits receptor activated PLC signalling leading to an altered transcriptional response and reduced neuronal excitability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

My AP Biology ThoughtsUnit 4 Cell Communication and Cell CycleWelcome to My AP Biology Thoughts podcast, my name is Nikki Evich and I am your host for episode #82 called Unit 4 Cell Communication and Cell Cycle: Intro to Signaling Transduction Pathway. Today we will be discussing the components that make up a pathway. Segment 1: Introduction to the signaling transduction pathway● Signal also call ligand binds to a receptor on target cell membrane ○ , starts the pathway, ○ had to fit receptor, ○ once bound, transduction is initiated ● Receptor- intracellular or extracellular ○ Binding domain recognizes specific chemical messengers ● transduction-lots of varian with transduction ○ Could activate inactive protein by phosphorylating ○ Amplifies with secondary messengers ● Response- what the end result is ○ Can be short or long ○ Activate enzyme or move cell-short ○ Alter gene expression levels or cell division (apoptosis)-long Segment 2: More About the signaling transduction pathway● Type 1 ● Once the food is broken down into glucose, these molecules are then absorbed into the bloodstream. The high glucose levels in the bloodstream activate the beta cells in the pancreas to start producing insulin. Insulin is a hormone created in the pancreas. In the pancreas, beta cells are present which are in charge of secreting the insulin into the bloodstream once they detect an increase in blood glucose. Insulin travels to three main destinations-muscle, fat, and liver cells. ● This is where the transduction pathway happens ● The insulin will then bind to the insulin receptors. The insulin receptors are made up of extracellular alpha subunits and transmembrane beta subunits. ● When the insulin binds to the extracellular alpha subunits, the beta subunits become activated and auto phosphorylate. This means that they phosphorylate themselves. ● This leads to the phosphorylation and activation of the IRS protein. The IRS protein is regulated and can be phosphorylated by PTEN. PTEN can regulate phosphorylation and activate IRS Isaforms by dephosphorylating IRS. Once IRS is activated, proteins including PI3K will bind to the IRS protein through their P85 subunit. ● The PI3K will then phosphorylate PIP2 to PIP3. When the PIP3 concentration increases, other proteins like PDK1 and AKT are recruited towards the plasma membrane. PIP3 activates PDK1 which then phosphorylates AKT. ● Cells have reservoirs of intracellular vesicles that contain GLUT4, a glucose transporter. So in order for glucose to be let into the cell the glucose transporters have to translocate to the plasma membrane. However, AS160 inhibits this process. ● Luckily, phosphorylated AKT inactivates AS160. So when AKT is phosphorylated by PDK1, AS160 is inactivated which in turn allows for the translocation of glut 4 so it can embed itself in the membrane. Now glucose can get into the cell for storage and other purposes. Segment 3: Connection to the Course● Involved in evolution-some cell transduction pathways stayed the same ○ Track common ancestors Interruptions in this pathway are serious like with the brain sending signals out ● Involved in negative feedback loops and homeostasis ● Body is constantly sending signals, though it may seem minute it makes you be able to do all you do ● In all walks of life ● Can be seen in all types of diseases and illness from Diabetes to cancer Thank you for listening to this episode of My AP Biology Thoughts. For more student-ran podcasts and digital content, make sure that you visit www.hvspn.com. Bye now! Music Credits: ● "Ice Flow" Kevin MacLeod (incompetech.com) ● Licensed under Creative Commons: By Attribution 4.0 License ● http://creativecommons.org/licenses/by/4.0/ Subscribe to our Podcast https://podcasts.apple.com/us/podcast/my-ap-biology-thoughts/id1549942575 (Apple Podcasts) https://open.spotify.com/show/1nH8Ft9c9f6dmo75V9imCk (Spotify)...

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.03.364851v1?rss=1 Authors: Lizarrondo, J., Klebl, D. P., Niebling, S., Abella, M., Schroer, M. A., Mertens, H. D. T., Veith, K., Svergun, D. I., Skruzny, M., Sobott, F., Muench, S., Garcia-Alai, M. M. Abstract: During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.30.361964v1?rss=1 Authors: Larsen, A. H., Sansom, M. Abstract: C2 domains facilitate protein-lipid interaction in cellular recognition and signalling processes. They possess a {beta}-sandwich structure, with either type I or type II topology. C2 domains can interact with anionic lipid bilayers in either a Ca2+-dependent or a Ca2+-independent manner. The mechanism of recognition of anionic lipids by Ca2+-independent C2 domains is incompletely understood. We have used molecular dynamics (MD) simulations to explore the membrane interactions of six Ca2+-independent C2 domains, from KIBRA, PI3KC2, RIM2, PTEN, SHIP2, and Smurf2. In coarse grained MD simulations these C2 domains bound to lipid bilayers, forming transient interactions with zwitterionic (phosphatidylcholine, PC) bilayers compared to long lived interactions with anionic bilayers also containing either phosphatidylserine (PS) or PS and phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back or side of the {beta} sandwich, whereas type II C2 domains bound canonically, via the top loops (as is typically the case for Ca2+-dependent C2 domains). C2 domains interacted strongly (up to 120 kJ/mol) with membranes containing PIP2 causing the bound anionic lipids to clustered around the protein. The C2 domains bound less strongly to anionic membranes without PIP2 (

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.14.338293v1?rss=1 Authors: Stefanski, K. M., Russell, C. M., Westerfield, J. M., Lamichhane, R., Barrera, F. N. Abstract: The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces anti-oncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using SMALPs to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TM are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions, and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.03.281378v1?rss=1 Authors: Pipatpolkai, T., Corey, R. A., Proks, P., Ashcroft, F. M., Stansfeld, P. J. Abstract: Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.22.216317v1?rss=1 Authors: Ma, N., Lee, S., Vaidehi, N. Abstract: Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in multi-component lipid bilayer. Using the multi-scale molecular dynamics simulations(50s), our comparative study between cell membrane and detergents shows that: the changes in inter-residue distances, known as activation microswitches, show an ensemble of states in the extent of activation in cell membrane. We forward a rheostat model of GPCR activation rather than a binary switch model. Phosphatidylinositol bisphosphate (PIP2) and calcium ions, through a tug of war, maintain a balance between the GPCR stability and activity in cell membrane. Due to the lack of receptor stiffening effects by PIP2, detergents promote more transitions among conformational states than cell membrane. These findings connect the chemistry of cell membrane lipids to receptor activity useful to design detergents mimicking cell membrane. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.22.164400v1?rss=1 Authors: Yao, C.-K., Li, T.-N., Chen, Y.-J., Wang, Y.-T., Lin, H.-C. Abstract: Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that the Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PIP2) compartmentalization governs such control. Strong stimuli trigger PIP2 microdomain formation at periactive zones. Upon exocytosis Flower translocates from SVs to periactive zones, where it increases PIP2 levels via Ca2+ influxes. Remarkably, PIP2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PIP2 microdomain compartmentalization. The PIP2 microdomains drive ADBE and SV reformation from bulk endosomes. PIP2 further sorts Flower to bulk endosomes, thereby terminating endocytosis. Hence, we propose that the interplay between Flower and PIP2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.18.158709v1?rss=1 Authors: Schechter, M., Atias, M., Abd Elhadi, S., Davidi, D., Gitler, D., Sharon, R. Abstract: alpha-Synuclein (a-Syn) is a protein implicated in the pathogenesis of Parkinson s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for a-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of a-Syn in these mechanisms is currently unclear. Here we have investigated the role of a-Syn in membrane trafficking through its association with acidic phosphoinositides (PIPs), such as phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and phosphatidylinositol 3,4-bisphosphate (PI3,4P2). Our results show that a-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor AP2 at clathrin-coated pits. Using endocytosis of transferrin, an indicator of clathrin mediated endocytosis (CME), we find that a-Syn involvement in endocytosis is specifically mediated through PI4,5P2 levels. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by a-Syn mutations. In accord with their effects on PI4,5P2 levels at the plasma membrane, the PD associated E46K and A53T mutations further enhance SV endocytosis. However, neither A30P mutation, nor Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of a-Syn, that interfere with phospholipid binding, affect SV endocytosis. This study provides evidence for a critical involvement of PIPs in a-Syn-mediated membrane trafficking. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.04.134544v1?rss=1 Authors: Mathiharan, Y. K., Glaaser, I. W., Zhao, Y., Robertson, M. J., Skiniotis, G., Slesinger, P. A. Abstract: G protein-gated inwardly rectifying potassium (GIRK) channels play important roles in controlling cellular excitability in the heart and brain. While structural data begin to unravel the molecular basis for G protein and alcohol dependent activation of GIRK channels, little is known about the modulation by cholesterol. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS), and PIP2. The structures and their comparison reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain (TMD). CHS potentiates the effects of PIP2, which stabilizes the inter-domain region and promotes the engagement of the cytoplasmic domain (CTD) onto the transmembrane region. The results suggest that CHS acts as a positive allosteric modulator and identify novel therapeutic sites for modulating GIRK channels in the brain. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.27.059600v1?rss=1 Authors: Maguire, E., Menzies, G. E., Phillips, T., Sasner, M., Williams, H. M., Czubala, M. A., Evans, N., Cope, E. L., Sims, R. C., Howell, G., Lloyd-Evans, E., Williams, J., Allen, N. D., Taylor, P. R. Abstract: Recent genome-wide association studies of Alzheimer's disease (AD) have identified variants implicating immune pathways in disease development. A rare coding variant of PLCG2, which encodes PLC{gamma}2, shows a significant protective effect for AD (rs72824905, P522R, P=5.38x10-10, Odds Ratio = 0.68). Molecular dynamic modelling of the PLC{gamma}2-R522 variant, situated within the auto-inhibitory domain of PLC{gamma}2, suggests a structural change to the protein. Through CRISPR-engineering we have generated novel PLCG2-R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model, neither of which exhibits alterations in endogenous PLCG2 expression. Mouse microglia and macrophages and hiPSC-derived microglia-like cells with the R522 mutation, all demonstrate a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This signalling alteration manifests as enhanced cellular Ca2+ store release (~20-40% increase) in response to physiologically-relevant stimuli (e.g. Fc receptor ligation and A{beta} oligomers). This hyperfunctionality resulted in increased PIP2 depletion in the cells with the PLC{gamma}2-R522 variant after exposure to stimuli and reduced basal detection of PIP2 levels in vivo. These PLC{gamma}2-R522 associated abnormalities resulted in impairments to phagocytosis (fungal and bacterial particles) and enhanced endocytosis (A{beta} oligomers and dextran). PLC{gamma}2 sits downstream of disease relevant pathways, such as TREM2 and CSF1R and alterations in its activity, direct impacts cell function, which in the context of the inherent drugability of enzymes such as PLC{gamma}2, raise the prospect of manipulation of PLC{gamma}2 as a therapeutic target in Alzheimer's Disease. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.23.057851v1?rss=1 Authors: Verma, P., Eaton, M., Kienle, A., Flockerzi, D., Yang, Y., Ramkrishna, D. Abstract: Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent, painful side effect which arises due to a number of chemotherapy agents. CIPN can have a prolonged effect on quality of life. Chemotherapy treatment is often reduced or stopped altogether because of the severe pain. Currently, there are no FDA-approved treatments for CIPN partially due to its complex pathogenesis in multiple pathways involving a variety of channels, specifically, voltage-gated ion channels. A surrogate of neuropathic pain in an in vitro setting is hyperexcitability in dorsal root ganglia (DRG) peripheral sensory neurons. Our study employs bifurcation theory to investigate the role of voltage-gated ion channels in inducing hyperexcitability as a consequence of spontaneous firing, due to the common chemotherapy agent paclitaxel. Our mathematical investigation suggests that the sodium channel Nav1.8 and the delayed rectifier potassium channel conductances are the most critical for hyperexcitability in normal firing small DRG neurons. Introducing paclitaxel into the model, our bifurcation analysis predicts that hyperexcitability is extreme for a medium dose of paclitaxel, which is validated by multi-electrode array recordings. Our findings using multi-electrode array experiments reveal that the Nav1.8 blocker A-803467 and the delayed rectifier potassium enhancer L-alpha-phosphatidyl-D-myo-inositol 4,5-diphosphate, dioctanoyl (PIP2) have a protective effect on the firing rate of DRG when administered separately together with paclitaxel as suggested by our bifurcation analysis. Copy rights belong to original authors. Visit the link for more info

Wed, 15 Apr 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18857/ https://edoc.ub.uni-muenchen.de/18857/1/Liu_Chen.pdf Liu, Chen ddc:540, ddc:500, Fakultät

Modification of a bound lipid modulates the activity of a nuclear receptor.

Adaptor subunits of phosphoinositide 3-kinase gamma specify distinct cellular responses.

Thu, 8 May 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12029/ https://edoc.ub.uni-muenchen.de/12029/1/Da_Ines_Olivier.pdf Da Ines, Olivier ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Podosomen sind ein prominenter Teil des Aktinzytoskelettes primärer humaner Makrophagen und wahrscheinlich essentiell für Adhäsion, Matrixverdau und gerichtete Migration. In der vorliegenden Arbeit wurde die Regulation dieser Strukturen untersucht. Es konnte zunächst gezeigt werden, dass Monozyten Podosomen nicht nur auf starren, künstlichen Oberflächen wie Glas-Deckgläschen ausbilden, sondern auch auf einem Monolayer aus Endothelzellen. Dies unterscheidet sie klar von anderen Adhäsionsstrukturen wie z.B. focal adhesions. Auch in verschiedenen Zelllinien, unter anderem in Krebszellen, ließen sich podosomale Strukturen nachweisen bzw. induzieren. Diese Befunde sind Hinweis einerseits auf die physiologische Relevanz von Podosomen und andererseits auf eine wahrscheinlich weite Verbreitung dieser Strukturen in verschiedenen Zelltypen. Podosomen sind hochdynamische Strukturen mit einer Halbwertszeit von 2-12 Minuten, das heißt, es werden permanent Podosomen abgebaut und neu gebildet. Dazu ist die Polymerisation und Depolymerisation von filamentösem (F-)Aktin notwendig. Regulationsmechanismen F-Aktin-aufbauender Wege sind gut untersucht und bekannt, weshalb in der vorliegenden Arbeit F-Aktin-abbauende Wege untersucht wurden. Ein wichtiger Regulator des Aktinzytoskelettes ist Cofilin, das die Depolymerisierung von Aktinfilamenten beschleunigt und unter anderem durch Phosphorylierung am Serin-3 inaktiviert werden kann. Folgende Ergebnisse sprechen für eine wichtige Rolle von Cofilin in der Podosomen-Regulation: Es konnte eine spezifische Lokalisation von Cofilin und phosphoryliertem Cofilin in der Aktin-reichen Podosomen-Kernstruktur nachgewiesen werden. Im Western Blot zeigte sich eine Korrelation des Grades der Cofilin-Phosphorylierung mit der Podosomenanzahl. Durch Mikroinjektion eines kurzen Peptids, welches die Cofilin-Phosphorylierung inhibiert, sowie durch Transfektion von Cofilin-siRNA konnte die Podosomen-Bildung reduziert werden. Die am besten untersuchten Cofilin-Kinasen sind die LIM-Kinasen 1 und 2. Mittels RT-PCR war in unserer Arbeitsgruppe bereits die Expression von LIMK1 in Makrophagen nachgewiesen worden. Auch Ergebnisse im Western Blot sowie in DNA-Arrays weisen auf LIMK1 als dominante Isoform in Makrophagen hin. In fixierten Präparaten konnte allerdings weder mit kommerziell erhältlichen noch mit einem selbst hergestellten, gegen die LIM-Domänen von LIMK1 gerichteten Antikörper eine spezifische Lokalisation von LIMK1 an Podosomen nachgewiesen werden. Mittels Nucleofection wurden deshalb verschiedene LIM-Kinase-Konstrukte transfiziert und überexprimiert. Dabei bestätigten sich die Ergebnisse der Antikörperfärbungen, keines der Konstrukte war in Podosomen zu finden. Alle Konstrukte mit Kinase-Aktivität führten zum raschen Krampfen und Ablösen der Zellen, wobei die Adhäsionsfläche bis zuletzt mit Podosomen bedeckt war. Im Gegensatz zu den Befunden aus der Transfektion war durch Mikroinjektion der konstitutiv aktiven Kinase-Domäne von LIMK1 eine deutliche Reduktion der Podosomen-Bildung zu erzielen. Hier können konzentrationsabhängige Effekte eine Rolle spielen. Als Gegenspieler der LIM-Kinasen wurden die Phosphatasen PP1 und PP2A beschrieben. Eine spezifische Lokalisation von PP2A an Podosomen war jedoch nicht nachzuweisen, zudem hatte eine Inhibition der beiden Phosphatasen keinen Effekt auf die Podosomenbildung oder den Podosomenabbau. Dies spricht gegen eine Beteiligung von PP1 oder PP2A an der Podosomenregulation. LIM-Kinasen selbst können durch Effektoren der Rho-GTPasen Rho, Rac und Cdc42 reguliert werden. So aktiviert der Rho-Effektor ROCK LIMK1 und LIMK2. Der ROCK-Inhibitor Y?27632 führte zu einer Störung der Podosomen-Verteilung, auch die Podosomen-Neubildung wurde stark inhibiert. Dies spricht für eine Beteiligung von ROCK an der Podosomenregulation. Auch Rac und Cdc42 können durch die gemeinsamen Effektoren der PAK-Familie eine Aktivierung von LIMK1 bewirken, dabei sind PAK1 und PAK4 die am besten untersuchten Isoformen. Die Transfektion verschiedener PAK1- und PAK4-Konstrukte führte jeweils zu einer Reduktion der Podosomen-Anzahl, unabhängig von der Kinase-Aktivität des Konstruktes. Die Kinase-inaktive PAK4-Mutante führte zu einer Reduktion des F-Aktin mit kleinen Podosomen, während die konstitutiv-aktive PAK4-Mutante große Podosomen mit vermehrtem F-Aktin bewirkte. Weitere Arbeiten zur Untersuchung vor allem von PAK4 in unserer Arbeitsgruppe konnten diese Ergebnisse bestätigen und quantifizieren sowie weitere Interaktionspartner nachweisen. Eine weitere Regulationsmöglichkeit von Cofilin ist die Bindung des second messengers PIP2, welcher unter anderem durch Isoformen der Phospholipase C (PLC) hydrolysiert werden kann. Die Mikroinjektion zweier Peptide, die laut Literatur zu einer PIP2-Inhibition bzw. einer Steigerung des PIP2-Abbaus führen, hatte keinen Einfluss auf Podosomen. Durch Transfektion der PH-Domäne von PLCd1, welche als PIP2-Sensor eingesetzt werden kann, konnte jedoch eine teilweise Lokalisation von PIP2 an Podosomen gefunden werden. Mit spezifischen Antikörpern konnte zudem eine Lokalisation von PLCb1 im Aktin-reichen Podosomenkern und von PLCb2 in der podosomalen Ringstruktur nachgewiesen werden, PLCb3 zeigte keine spezifische Lokalisation. Auch ein PLCb2-Konstrukt reicherte sich nach Transfektion in der podosomalen Ringstruktur an. Der PLC-Inhibitor U-73122 führte zu einem kompletten Verschwinden der Podosomen mit nachfolgender Ablösung der Zellen. Aufgrund dieses Befundes und der spezifischen Lokalisation ist von einer Beteiligung der PLCb1 und PLCb2 in der Podosomen-Regulation auszugehen. Im Rahmen der vorliegenden Arbeit konnten somit wichtige Effektoren der podosomalen Aktinregulation identifiziert werden: Cofilin als direkter Interaktionspartner von Aktin, LIMK1 als Cofilin-Regulator sowie ROCK und PAK als upstream-Regulatoren in der Signalkaskade. Darüber hinaus scheinen PLCb1 und PLCb2, möglicherweise über PIP2, ebenfalls an der Podosomen-Regulation beteiligt zu sein. Dies legt die Grundlage für weitere Untersuchungen über die molekularen Mechanismen der podosomalen Aktinregulation.

Im Rahmen dieser Arbeit wurde mit Fluoreszenz-Korrelationsspektroskopie (FCS) zum ersten Mal systematisch die Bindung von fluoreszenzmarkierten Peptiden an anionischen Lipidmembranen untersucht. Mit dieser Methode konnten Einzelmolekül-Messungen zur Bindung von myristoyliertem Alanin-reichen C Kinase Substrat, MARCKS (151-175), an unilamellare Vesikel mit einem Durchmesser von 100 nm durchgeführt werden. Die Vesikel bestanden aus dem neutralen Lipid Phosphatidylcholine (PC) und den negativ geladenen Lipiden Phosphatidylserine (PS) oder Phosphatidylinositol 4,5-bisphosphate (PIP2). Eine Signal/Rausch-Analyse ermöglichte die Bestimmung der Sensitivität und des linearen Messbereichs der Methode. Auf Grund der unterschiedlichen Korrelationszeiten der freien und gebundenen Peptide folgte aus den gemessenen Autokorrelationskurven der prozentuale Anteil des gebundenen Pepids. Die Bindung von MARCKS(151-175) an die anionischen Vesikel wurde für verschiedene prozentuale Anteile von PS und PIP2 gemessen. Sie war umso stärker, je höher der Anteil anionischer Lipide in der Membran und damit die attraktive elekrostatische Wechselwirkung war. Die ermittelten Bindungskonstanten stimmten gut mit den Resultaten überein, die mit etablierten konventionellen Techniken wie NMR, ITC oder Spinmarkierung gewonnen wurden. Die Experimente konnten zeigen, dass mit FCS direkte Messungen von nanomolaren Peptidkonzentrationen möglich sind. FCS stellt eine präzise Methode zur Untersuchung der Wechselwirkung von Peptiden und Proteinen mit Lipidmembranen dar. Im zweiten Teil der Arbeit wurde die Selbstorganisation und das Aggregationsverhalten von verschiedenen synthetischen Gentransfer-Komplexen studiert. Mit der Methode der quantitativen Fluoreszenzmikroskopie wurde die Größenverteilung bestimmt und die Zahl der Plasmide pro Gentransfer-Komplex berechnet. Die Polyplexe stellen im Unterschied zu den Lipoplexen unter den experimentellen Bedingungen ein polydisperses kolloidales System dar. Unter dem Einfluss eines natürlichen Surfactants (Alveofact) war ein umgekehrtes Verhalten zu beobachten. Die Messungen zur Kinetik des kolloidalen Systems erfolgten mit der Fluoreszenz-Korrelationsspektroskopie. Mit dieser Methode wurde der Einfluss der Ionenkonzentration und von Alveofact auf die Aggregationskinetik von verschiedenen positiv geladenen Gentransfer-Komplexen untersucht. Die Experimente zeigten, dass die Geschwindigkeit der kolloidalen Aggregation mit der Ionenkonzentration drastisch zunimmt und die mittlere Zahl der Plasmide pro Komplex als Funktion der Zeit linear ansteigt. Nach der Inkubation mit Alveofact stellten die Polyplexe auch unter physiologischen Bedingungen ein stabiles kolloidales System dar und bestanden im Mittel aus 3-5 Plasmiden. Auch in diesem Fall wurde bei den DNA/Lipofectamine-Komplexen ein anderes Aggregationsverhalten beobachtet. Sie bildeten ohne die Einwirkung von Alveofact unabhängig von der Ionenkonzentration ein stabiles Kolloid und bestanden im Mittel aus nur zwei kondensierten Plasmiden. Dagegen führte die Inkubation mit Alveofact zu einer langsamen kolloidalen Aggregation. Die Lipoplexe erreichten in diesem Fall nach 60 min eine Größe von ~3-4 Plasmiden pro Komplex. Mit Fluoreszenz-Korrelationsspektroskopie wurde das Transportverhalten verschiedener Vesikel und Gentransfer-Komplexe in einem negativ geladenen Polymer-Netzwerk (Mucin) untersucht. Die Größe und die Ladung der Partikel bestimmten die Diffusion durch das Polymer-Netzwerk. Kleinere Durchmesser und höhere negative Ladungen der Vesikel trugen zu einem effektiveren Transport durch das Netzwerk bei. Aus der Diffusionskonstante und der makroskopischen Viskosität der Polymerlösung wurden die Abweichungen von der Stokes-Einstein-Relation berechnet. Das Diffusionsverhalten anionischer Vesikel wurde zum Vergleich auch in einem positiv geladenen Kollagen-Netzwerk studiert. Bei den synthetischen Gentransfer-Komplexen mit positiver Überschussladung wurde auf Grund der Bindung zum Netzwerk ein deutlicher Abfall der Diffusionskonstante als Funktion der Polymer-Konzentration beobachtet. Im Unterschied dazu zeichneten sich die negativ geladenen Komplexe wegen der repulsiven elektrostatischen Wechselwirkung durch einen effektiveren Transport im Mucin-Netzwerk aus. Unter dem Einfluss von Alveofact zeigten die positiv geladenen Komplexe ein ähnliches Transportverhalten wie die negativ geladenen Komplexe. Eine gezielte Beschichtung der Komplexe ermöglichte also einen verbesserten Transport durch das Polymer-Netzwerk, was im Zusammenhang mit einem effizienten Gentransfer von Interesse ist.

The reaction product of phospholipase D (PLD), phosphatidic acid (PA), was found to stimulate phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity in vitro. In the present study, we have examined wether PLD affects the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led to an increase in PIP-5-kinase activity and to elevated PIP2 levels in intact cells. As both PLD and PIP-5-kinase are stimulated by the GTPases Arf1 and RhoA, we investigated in the following, if PLD is involved in the regulation of PIP2 synthesis by these GTPases. Both PLD1- and PLD2-induced PIP2 synthesis was completely blocked by coexpression of catalytically inactive Arf1 T31N. Reversely, the effect of constitutive active Arf1 Q71L was fully inhibited by catalytically inactive PLD constructs. Whereas the effects of Arf1 Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L with wild-type PLD1 led to a synergistic increase in PIP-5-kinase activity. Previously, we have shown that RhoA regulates the activity of PLD and PIP-5-kinase by its downstream effector Rho-kinase. Expression of small amounts of inactive PLD1, but not of PLD2, nearly completely abolished Rho-kinase-stimulated PIP-5-kinase activity. Also expression of a non-phosphorylatable mutant of cofilin, which participates in the signalling cascade from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the stimulating effect of Rho-kinase on PIP2 synthesis. These findings suggest that PLD1 is involved in the stimulation of PIP-5-kinase by Arf1 as well as by RhoA and Rho-kinase. After sucrose density gradient centrifugation of HEK-293 cell lysates, we isolated two separate PIP2 pools. PLD1 and Arf1 selectively control the non-caveolar PIP2 pool in the high density fraction, whereas PLD2 affected PIP2 in both pools. In summary, these data suggest that particularly PLD1, apparently by the production of PA, functions as a physiological regulator of PIP-5-kinase that controls the synthesis of cellular PIP2 downstream to Arf1 and RhoA.

MIPs (major intrinsic proteins) sind eine Gruppe von Transport-Proteinen, die ubiquitär in Archaea, Pro- und Eukaryoten zu finden sind. Neben spezifischen Wasserkanalproteinen (Aquaporine) sind einige Mitglieder dieser Familie permeabel für andere kleine und ungeladene Moleküle, wie z.B. Glycerin. Als Grundlage zur Funktionsaufklärung der 38 MIP-Mitglieder in A. thaliana wurden ihre transkriptionellen Reaktionen untersucht. Dazu wurde ein DNA-Array mit Sonden aus dem 3´-untranslatierten Bereich dieser Gene entwickelt, der die Unterscheidung der oft hoch homologen Mitglieder auf Transkript-Ebene zuließ, wozu längere cDNA-Sonden nicht geeignet sind. Eine TIP1;2 cDNA-Sonde, die Teile der kodierenden Sequenzen enthielt, zeigte in einem Hybridisierungsexperiment eine 40 %-ige Kreuzhybridisierung mit dem homologen TIP1;1. Die Spezifität der Sonden wurde in Zusammenarbeit mit dem Munich Information Center for Protein Sequences bioinformatisch überprüft. Das stringente Auswahlkriterium dieser Analysen, woraufhin eine Sonde bis zu einer 70 %-igen Homologie über 70 bp nicht kreuzhybridisiert, konnte auch experimentell gestützt werden. Der Vergleich der Signalintensitäten einer 172 bp langen, spezifischen Sonde mit einer 774 bp langen cDNA-Sonde ließ zudem darauf schließen, dass die Spezifität der verwendeten 3´-UTR-Sonden die Sensitivität nicht beeinträchtigte. Eine Organ-spezifische Expressions-Analyse zeigte, dass MIPs in allen untersuchten Organen (Wurzeln, Blätter, Stängeln, Blüten und Schoten) exprimiert werden. Die meisten MIPs (24 von 38) und die höchsten Expressionsniveaus wurden in der Wurzel detektiert. In Blättern konnten hingegen nur 11 von 38 MIP-Mitgliedern nachgewiesen werden. Die geringsten Expressionen zeigten die Mitglieder aus der NIP- und SIP-Subfamilie. Zu den am höchsten und in der Pflanze ubiquitär exprimierten MIPs zählten die PIP-Mitglieder PIP1;1, PIP1;2 und PIP2;1 sowie die TIP-Mitglieder TIP1;1 und TIP2;1, von denen bekannt ist, dass sie als Wasserkanal-Proteine fungieren. Die Möglichkeit, die Wasserpermeabilität von Membranen regulieren zu können, dürfte somit von zentraler Bedeutung in der ganzen Pflanze sein. Neben ubiquitär exprimierten MIPs sind einige nur ausschließlich oder bevorzugt in bestimmten Organen zu finden, z.B. PIP1;4, PIP2;6 und PIP2;7 in der Blüte und NIPs hauptsächlich in der Wurzel. Deren Funktion könnte neben einem Organ- oder Zell-spezifischen Wassertransport auch die Permeation anderer ungeladener Moleküle beinhalten, da die Transporteigenschaften dieser Mitglieder nicht geklärt sind. Das Expressionsprofil der MIP-Genfamilie in verschiedenen Organen sowie die unterschiedliche Reaktion auf Wasserstress (s.u.) lassen auf differenzielle Funktionen der einzelnen MIP-Mitglieder schließen. Lediglich TIP1;1 und TIP1;2 konnten nach diesen Kriterien nicht eindeutig unterschieden werden. Durch Zugabe von 100 mM NaCl und 200 mM Sorbiotol zu hydroponisch angezogenen A. thaliana-Pflanzen wurde über einen Zeitraum von 48 Stunden die transkriptionelle Reaktion der MIP-Familie auf diese Wasserstressbedingungen verfolgt. In Wurzeln wurde festgestellt, dass innerhalb der ersten 24 Stunden bei beiden Stressoren die hoch exprimierten PIP-Aquaporine PIP1;1 und PIP2;2 sowie PIP1;3 reprimiert, TIPAquaporine wie TIP1;1 und TIP2;1 zu diesem Zeitpunkt jedoch unbeeinflusst sind. Die Pflanze verringert demnach bei Wasserstress zuerst die Wasserpermeabilität der Plasmamembran, sofern sich die transkriptionelle Suppression auf Proteinebene widerspiegelt. Interessanterweise zeigte sich im Blatt eine frühe Repression der hoch exprimierten TIPAquaporine TIP1;1 und TIP2;1. Die in der Wurzel reagierenden Aquaporine aus der PIPSubfamilie zeigen im Blatt jedoch keine transkriptionellen Änderungen. Zusammenfassung 95 Diese frühen Repressionen von TIP1;1 und TIP2;1 lassen vermuten, dass es in Blättern wichtig ist, möglichst rasch unter Wasserstress die Wasserpermeabilität des Tonoplasten zu senken. Dies könnte der Stabilisierung des Zell-Turgors dienen und/oder mit einem verringerten Blattwachstum einhergehen. Die ähnlichen Transkriptionsantworten und Kinetiken der MIPs bei NaCl- und Sorbitolstress lassen zudem vermuten, dass MIPs auch unter NaCl-Stress primär auf die osmotische Veränderung in der Nährlösung reagieren bzw. über ähnliche Signalwege reguliert werden. Die parallele Untersuchung transkriptioneller Änderungen von bekannten Stress-Markergenen und Genen des Sekundärmetabolismus unter NaCl- und Sorbitolstress ergab zusätzliche Hinweise auf überlappende und Stressor-spezifische Reaktionen in Wurzeln und Blättern. Eine bekannte Reaktion auf Salz- und osmotischen Stress ist die Bildung von reaktiven Sauerstoff-Spezies, die sowohl als Signalmoleküle fungieren als auch Schädigungen verursachen können. Die Induktionen der beiden Peroxidasen GPX1 und PRXCB in Blättern und Wurzeln deuten auf eine Beteiligung bei Entgiftungsreaktionen von H2O2 unter Wasserstress hin. Einzelne Mitglieder aus der Familie der UDP-Glycosyltransferasen und Cytochrom P450-Monooxygenasen, wie UGT74F2 und CYP81D1, zeigten bei Salz- und Sorbitolstress überlappende Reaktionen, was darauf hindeutet, dass spezifische Teile des Sekundärstoffmetabolismus ähnlich beeinflusst werden. Daneben zeigten andere Mitglieder aus diesen Gen-Familien spezifische Reaktionen auf die beiden Stressoren. So waren in der Wurzel nach 48 Stunden Sorbitolstress viele CYPs und UGTs reprimiert. Die Pflanze scheint also exklusiv bei Sorbitolstress spezifische, in ihrer genauen Funktion noch unbekannte Teile des Sekundärmetabolismus zu supprimieren. Die unterschiedlichen Reaktionen einer Reihe weiterer Gene auf Salz oder Sorbitol in Blättern und Wurzeln identifizierten zudem differenzielle Stress-Antworten innerhalb der Pflanze. Es wurden zwei Insertionsmutanten in den MIP-Genen PIP2;1 und PIP1;4 isoliert. Transkript-Untersuchungen dieser Mutanten zeigten, dass durch das Ausschalten dieser PIPs alle anderen MIP-Mitglieder, sowohl bei ungestressten als auch unter NaCl-Stress- Bedingungen, keine Änderungen in ihrer Transkriptionsantwort im Vergleich zum Wildtyp zeigen. Möglicherweise besitzen die untersuchten PIP-Mitglieder eine spezifische Funktion, die andere MIPs nicht kompensieren können, oder möglicherweise kommt es in der Pflanze zu veränderten Reaktionen, die anhand der vorliegenden Untersuchungen nicht erkennbar waren.

A large number of actin-binding proteins regulates the dynamics of the actin cytoskeleton. Here we report the identification and characterization of two new proteins in Dictyostelium discoideum: the novel cytoskeleton protein villidin and a third profilin isoform. One goal of the project was to analyze the features of the villidin sequence in detail and to investigate the protein by molecular, biochemical and cell biological approaches. Villidin has a calculated molecular mass of 190,000 Da. Based on the domain structure of the protein, villidin can be assigned to the gelsolin/villin-family as well as to the WD-repeat family. In principal, the group of the WD-repeat proteins includes regulatory proteins which are involved in signal transduction and other important cellular processes. The N-terminus of villidin harbours between four and eight of the specific WD-repeats and forms probably a β-propeller structure. The WD-domain is followed by an intervening domain of 400 amino acids that leads to the second characteristic villidin domain at the C-terminus. This part of the sequence exhibits a similarity to villin, though the first of the six villin domains is absent in villidin. However the typical headpiece is present. Villidin mRNA and protein are expressed in low amounts during growth and early aggregation, they are increased during development and reach highest levels at the tipped aggregate stage. The protein is present in the cytosol as well as in the cytoskeletal and the membrane fraction. These biochemical results are in agreement with the immunofluorescence data. The endogenous villidin is homogeneously distributed throughout the cytosol and localizes at vesicular structures. Colocalization experiments lead to the assumption that these structures might belong to a still unknown population of vesicles. GFP fusion proteins with the villin homology domains show a similar distribution, whereas GFP fusions of the N-terminal part encompassing the WD-repeats are present in F-actin rich regions at the plasma membrane and on internal membranes during motility, pinocytosis and phagocytosis. Mutants lacking villidin do not show an aberrant phenotype during growth and development but are defective in motility and phototaxis in the multicellular slug stage. Based on this defect and the multidomain structure of the protein, villidin might be involved in signal transduction processes leading to phototactic movement. The second part of the project dealt with a new gene which codes for a third profilin isoform in D. discoideum. The well-known Dictyostelium profilin isoforms I and II are able to interact with G-actin, PIP2 and poly-L-prolin. As in the case of profilin I and II, profilin III is encoded by a single gene. In contrast to profilin I and II, the transcription of profilin III is not developmentally regulated. All three isoforms show the typical limited homology at the amino acid level. The recombinant Profilin III protein cosediments with poly-L-prolin, inhibits the actin-polymerisation and the PIP2 interaction of profilin III competes with the G-actin affinity. The low expression of Profilin III mRNA in growing and developing cells suggests a distinct role of profilin III because a low protein concentration argues against an actin sequestering function.