Podcasts about tirf

Dr. Carr is a Professor of Geriatric Medicine in the Department of Medicine and Neurology at Washington University at St. Louis. Dr. Carr is a clinician in the Memory Diagnostic Center and Geriatric Assessment Clinic at WU where he maintains an outpatient consulting practice in dementia and geriatric care. He is involved in Alzheimer's Disease drug trials and was the site PI in the GRADUATE trial and the Green Memory Trial in the WU Alzheimer's Treatment Unit. He has been and/or currently is a principal investigator or co-investigator on research, national guidelines, and/or educational initiatives related to Alzheimer's and/or medical conditions and driving with funding through NIA, NEI, and Missouri Department of Transportation. He also has been active in consulting work with NHTSA, TIRF, University of Toronto, Medscape, UpToDate and the American Geriatric Society. He has been the primary author or co-author on over 100 peer review manuscripts and chapters, most related to older drivers. In this episode we discuss:How to reduce risk of alzheimersWhy you should care about azlheimers in your 30sHow much alcohol is safe for your brainHow to improve your memory for the long termThis episode is brought to you by Timeline Nutrition, PaleoValley, Inside Tracker, 1stPhorm Join me at the Forever Strong Summit on January 13-14 for a transformative in-person experience, where I will share cutting-edge insights on optimizing health, performance, and longevity, empowering you to unlock your full potential. Don't miss this exclusive opportunity to learn from renowned experts in the field – secure your spot at https://drgabriellelyon.com/forever-strong-summit/Order Dr. Lyon's Book Forever Strong - https://drgabriellelyon.com/forever-strong/Mentioned in this episode:15% Off Your Purchasehttps://paleovalley.com/drlyon10% off your first order of Mitopurehttps://timelinenutrition.com/DRLYONVisit 1st Phorm Website for Free Shipping on orders $75+http://www.1stphorm.com/drlyonInside Tracker 20% Off the Entire Storehttps://info.insidetracker.com/drlyon

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548819v1?rss=1 Authors: Warner, H. M., Chen, T., Mahajan, S., ter Beest, M., Linders, P., Franciosa, G., Bianchi, F., van den Bogaart, G. Abstract: The mounting of an adaptive immune response is critical for removing pathogens from the body and generating immunological memory. Central to this process are myeloid cells, which sense pathogens through a variety of cell surface receptors, engulf and destroy pathogens and become activated. Activation is essential for the release of cytokines as well as the cell-surface presentation of pathogen-derived-antigens. Activation-induced cytokine release by myeloid cells requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is becoming increasingly clear that trafficking machinery has to be re-programmed through post-translational modifications to dynamically regulate cytokine secretory events. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term stimulation with the pathogenic stimulus lipopolysaccharide (LPS) is sufficient to up-regulate IL-6 secretion rates in human blood monocyte-derived dendritic cells and that this secretion is asymmetric and thus polarised. Using bioinformatics analysis of our phosphoproteomic data, we demonstrate that LPS stimulation of monocyte-derived dendritic cells rapidly reprograms SNARE-associated membrane trafficking machinery, through phosphorylation/dephosphorylation events. Finally, we link this enhanced rate of secretion to the phosphorylation of the SNARE protein VAMP3 at serine 44 (48 in mice), by showing that this phosphorylation drives the release of VAMP3 by its chaperone WDFY2 and the complexing of VAMP3 with STX4 at the plasma membrane. 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/2023.07.02.547398v1?rss=1 Authors: Yang, J., Ran, K., Ma, W., Chen, L., Chen, C., Zhang, C. M., Ye, H., Lu, Y. M., Ran, C. Abstract: Reduction of the production of amyloid beta (A{beta}) species has been intensively investigated as a potential therapeutic approach for Alzheimer disease (AD). However, the degradation of A{beta} species, another potentially beneficial approach, has been far less explored. In this study, we discovered that ceruloplasmin (CP), an important multi-copper oxidase (MCO) in human blood, could degrade A{beta} peptides. We also found that the presence of Vitamin C could enhance the degrading effect in a concentration-dependent manner. We then validated the CP-A{beta} interaction using total internal reflection fluorescence (TIRF) microscopy, fluorescence photometer, and fluorescence polarization measurement. Based on the above discovery, we hypothesized that other MCOs had similar A{beta}-degrading functions. Indeed, we found that other MCOs could induce A{beta} degradation as well. Remarkably, we revealed that ascorbate oxidase (AO) had the strongest degrading effect among the tested MCOs. Using induced pluripotent stem (iPS) neuron cells, we observed that AO could rescue neuron toxicity which induced by A{beta} oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an A{beta}-induced deficit in synaptic transmission in the hippocampus. To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment. 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/2023.05.05.539649v1?rss=1 Authors: Morales, E. A., Tyska, M. J. Abstract: Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. Using in vitro TIRF microscopy assays, we found that MISP exhibits a clear binding preference for filaments enriched in ADP-actin monomers. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions. 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/2023.05.04.539490v1?rss=1 Authors: Pimm, M. L., Marcin, A. G., Haarer, B. K., Eligio, M. A., Henty-Ridilla, J. L. Abstract: Cell processes require precise regulation of actin polymerization at filament plus ends to execute normal functions. The detailed mechanisms used to control filament assembly at plus ends in the presence of diverse and often opposing regulators is not clear. Here we explore and identify residues important for the plus-end related activities of IQGAP1. In multi-wavelength TIRF assays, we directly visualize dimers of IQGAP1, mDia1, and capping protein (CP) on filament ends alone and as a multicomponent end binding complex. IQGAP1 promotes the turnover of end-binding proteins, reducing the dwell times of CP, mDia1, or mDia1-CP "decision complexes" by 8-18-fold. Loss of these activities in cells disrupts actin filament arrays, morphology, and migration. Together, our results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how actin assembly is regulated in cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

With the Arrows on a bye week, Stu and Derek breakdown the biggest stories from week 3 in MLR action and the Vancouver stop on the Sevens Series. Then they are joined by Arrows' President and General Partner Bill Webb and Vice President and General Manager Tim Matthews. It's a deep dive into the business side of the Arrows including big off-season moves, the Hangar, plans for the academy sides, the partnership with TIRF, what fans can expect from the Arrows' match day experience, MLR expansion, and more!

President, Owner and General Partner of the Toronto Arrows, Bill Webb joins Game Play to discuss the Toronto Arrows Rugby Football Club, the expectations and goals for the upcoming season and the historical partnership with Toronto Inner-City Rugby Foundation, TIRF, a registered non-profit founded in 2011, builds community through rugby in 31 of Toronto's neighbourhoods.

In the second hour of Game Play, Matt Cauz is joined by President, Owner and General Partner of the Toronto Arrows, Bill Webb, to discuss TIRF, a local community impact organization, non-profit branding on jerseys and the rise of rugby in Canada. Then Matt talks to The Athletic senior baseball writer Keith Law about the start of Spring Training and the Blue Jays' season outlook.

In this episode, we chat with Robyn Robertson, the President and CEO of The Traffic Injury Research Foundation (TIRF) - a Canadian-based foundation that works with governments, communities, industries, and road safety organizations to make roads safer and reduce road deaths and injuries. TIRF has worked to improve road safety across Canada by generating and sharing knowledge on current and emerging issues and trends that affect road safety. What you'll learn: Behavioural change, attitudes, and the hurdles facing road safety professionals Researchers VS practitioners and why partnerships are important Canadian road safety statistics and trends How to minimise your risk of injury and death as a driver, passenger, or road-side user

Welcome to series 10 of Shoot Edit Chat Repeat, the podcast for people photographers. Hosted by two leading UK portrait photographers, Vicki Knights & Eddie Judd.     We're very happy to be back! In this first episode of series 10 we are chatting with documentary family photographer, Emma Collins.       Emma has been working as a family photographer since 2012 - she started as a family lifestyle photographer and we chat to her about the transition into a more documentary style.       Emma has acquired several top awards at the Documentary Family Awards, she was selected for the Female Voice Exhibition 2019, has been named top UK storyteller photographer 2020 by TIRF and has recently been shortlisted for the World Photography Organisation Sony Alpha Female Award 2021 and the British Photography Awards 2021.       Emma is also co-founder of Made for Documentary, a community hub providing expertise and education, on a mission to help documentary family photographers develop their skills and elevate the genre in the UK and beyond.       Here are some of the things we chat through:     - how to know if the style of photography you're shooting in is right for you   - how a documentary style family shoot works   - how to price a documentary family shoot   And lots more! Emma's website   Follow Emma on Instagram   Made For Documentary website   Follow Made for Documentary on Instagram   Facebook group: Made for Documentary. The Community         Repeat Recs for this week   Vicki - Glennon Doyle book Untamed and podcast 'We can do hard things'   Eddie - 'Pieces of Britney' podcast with Pandora Sykes   If you join us as a patron, for just $5 a month, you'll get instant access to to the extra juicy questions about pricing that we put to Emma. Click here for all of the details and to get instant access to the longer version of this episode, on your favourite podcast app.    We'd love to know what you think of this episode. Please leave us a review if you've enjoyed it, it helps other people to find the podcast.We'd love to know what you think of this episode. Please leave us a review if you've enjoyed it, it helps other people to find the podcast.   Email:  hello@shooteditchatrepeat.com   Instagram   Facebook   Thank you to The Design Space for sponsoring this episode. Click here for lots of free information about how to be the boss of your own website and for 20% off all their products. And thanks to Studio Ninja for their support of this episode. The most user-friendly studio management software on the market. Use the code 'studioninja50' at checkout to get a massive 50% off your first year's subscription.    Thank you to Shootproof online galleries for sponsoring Repeat Recs for this series. Get 25% off your first 12 months subscription by using the code SHOOTEDITCHATREPEAT.       Did you know we have an online course, The Art and Business of Personal Brand Photography. If you would love to succeed in this fast-growing genre, check it out here. You can get a 10% discount by using the code 'SHOOTEDIT'   Eddie Judd's Lightroom 121 virtual training     Get on the waitlist for Vicki's 7 week group training programme for family photographers. Delight & Dream starting again in Jan 2022  

A bit of a different Podcast episode this week, as I usually speak to a brilliant photographer, but today I have an incredibly average one... myself! Yes, it's Alan Law here, founder of This is Reportage and This is Reportage: Family. I just thought I'd try a solo episode - as I have various things I would like to say about the year; my personal experiences as a photographer myself, but also some behind-the-scenes thoughts and musings on running TiR and TiRF, and about the Podcast itself as well. I also have lots of thank yous to say! So if you're interested in my thoughts on all of that (and more), and think you can put up with only hearing my voice (in just one long take, amazingly!) then perhaps you'll enjoy it - but I also expect (of course!) that you may much prefer to hear the interviews, so don't worry, our usual interview format will be swiftly returning for all our future episodes! Listen at https://thisisreportage.com/podcast-episode-58-thoughts-on-2020/ Submissions are now open for our first Collections of 2021; a whole new Awards year, so if you’re wanting to get on our Top 100 Photographers list, you’ll have the best chance by entering our first Awards of 2021. Deadline for submissions is 23:59 GMT on 24th January 2021, for both our sites (This is Reportage & This is Reportage: Family), and submitted images can be taken from any time. Members receive 60 Individual Award and 18 Story Award entries per year, an unlimited number of images on your profile, exclusive educational videos, and many more benefits. Join us to submit at: This is Reportage (weddings) https://thisisreportage.com/ This is Reportage: Family (family) https://thisisreportagefamily.com/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.319400v1?rss=1 Authors: Hurley, M. E., Sheard, T. M. D., Norman, R., Kirton, H. M., Shah, S. S., Pervolaraki, E., Yang, Z., Gamper, N., White, E., Steele, D., Jayasinghe, I. Abstract: Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.17.254821v1?rss=1 Authors: Li, F., Sundaram, V. K., Gatta, A. T., Coleman, J., Krishnakumar, S., Pincet, F., Rothman, J. E. Abstract: Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture. Copy rights belong to original authors. Visit the link for more info

听众朋友你好，欢迎收听浏览加广中文台的周末网络广播节目。在今天的节目时间里，我们为您选播一个星期以来的几篇报道。 欢迎网友和听友们发表评论和看法。我们的电子信箱是：China@rcinet.ca 我们的新浪微博是：“加拿大国际广播-中文”; 我们的网站是： www.rcinet.ca. 每周五北美东部时间上午9点半，我们会有脸书直播（Facebook Live）。我们的Facebook： 加拿大国际广播 – 中文频道。 如果您想快捷、方便并全面地了解加拿大正在发生的事情，请下载安装加拿大国际广播App。请在安卓和苹果应用商店搜索关键词“加拿大国际广播”。 收听CH_Report_1-20191101-WRC10  本周报道精选 加拿大的石油钻探公司正在纷纷南迁至美国（亚明） 正在南迁中的加拿大石油公司之一 Citadel Drilling (Kyle Bakx/CBC) 加拿大西部石油产区的经济正在持续低迷，以至于很多石油公司为了生存，不得不花费巨额成本将采油业务迁到美国。在2014年油价暴跌之前，加拿大石油产区约有900座钻机， 而现在还剩约550座，其中也就有一半可开足马力。 -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- 魁北克省大麻新法规法定年龄提高到21岁, 禁售含大麻糖果和甜点（黎黎） (Martin Ouellet-Diotte/AFP/Getty Images) 魁北克省推出了针对大麻的2号法案，把消费和购买休闲大麻的法定年龄从18 岁提高到 21 岁，并禁售含大麻的糖果和甜点。新法规于 2020 年 1 月 1 日生效后，魁北克将成为加拿大在大麻问题上控制最严格的一个省份。 -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- 沈二：谷歌最新搜索更新 - 利用AI理解搜索背后的意义 -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- 爱德华王子岛12岁男孩出版讲民主政治的童话书（亚明) 12岁的Nathan MacNevin拿着他的新书， 后排自左至右为绿党省议员 Karla Bernard, 自由党临时党领 Sonny Gallant以及现任省长 Dennis King. (Sarah Keaveny-Vos/CBC) 加拿大的全国大选刚刚尘埃落定。还没到投票年龄的孩子们并不只是旁观者。爱德华王子岛的一名小学生以童话的形式出了一本书，向同学们讲述民主选举制度。这名小学生名叫内森•麦克奈文（Nathan MacNevin）。12岁的他已经认识到生活在民主国家中有多么重要。他说：“我相信孩子们应该从小就学习政治，它很有趣，我很喜欢”。 -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- 分心驾驶：导致致命车祸的重大因素（黎黎） (CBC) 根据加拿大安大略省交通伤害研究基金会（ Traffic Injury Research Foundation ，TIRF）的统计数据，在加拿大的某些地区，分心驾驶造成的死亡人数已经超过了由酒后或吸毒后驾驶造成的死亡人数。仅在安大略省进行一项调查表明，今年到目前为止，安省因分心驾驶导致的死亡人数为 44 人，因酒精或吸毒驾驶造成的死亡人数为 34 人。 一周图片报道 window.jQuery || document.write('

    A taste of this high yield original podcast that will definitely get you thinking For the full version, join PainExam.com or subscribe via the app Remember if you want board review questions, go to painexam (not the app) for full access Subscribe to our mailing list * indicates required Email Address *       DISCLAIMER: Doctor Rosenblum IS HERE SOLELY TO EDUCATE, AND YOU ARE SOLELY RESPONSIBLE FOR ALL YOUR DECISIONS AND ACTIONS IN RESPONSE TO ANY INFORMATION CONTAINED HEREIN. This podcasts is not intended as a substitute for the medical advice of physician to a particular patient or specific ailment.  You should regularly consult a physician in matters relating to yours or another's health.  You understand that this podcast is not intended as a substitute for consultation with a licensed medical professional.    Copyright © 2017 QBazaar.com, LLC  All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, recording or otherwise, without the prior written permission of the author.    

Although thermophoresis, i.e. the directed movement of molecules in a temperature gradient, was discovered more than 150 years ago, its molecular origin is not jet fully understood. Nonetheless thermophoresis is used as a principle in biomolecular binding measurements. Both topics are interesting and worth a scientific discussion. In this thesis, systematic experiments over a large parameter space were conducted. From these measurements a combination of different theories about its molecular origin could be verified. Thus, the first result of this thesis is that the phenomenon thermophoresis consists of different additive contributions. Some of them relate to the ionic nature of the molecule and are non-existent when the molecule is electrically neutral. The microscopic mechanism of these ionic contributions to thermophoresis is discussed in the first part. It continues the work on the capacitor model and explains a further contribution, which we call Seebeck effect in analogy to solid state physics. Through the different contributions we bridge the gap between local thermodynamic equilibrium approaches and non-equilibrium theories. Several applications will greatly benefit from understanding the molecular physics of thermophoresis. Pharmacological screens are conducted to determine the binding affinity of a whole molecular library to a target molecule and thus to identify the best candidates for a new drug. These screens will be improved when thermophoresis can be predicted and for example the influence of the buffer can be determined. Binding measurements of biomolecules can already be conducted in cell lysate. The second part of this thesis will show thermophoresis measurements inside living cells for the first time. This paves the way for in vivo binding measurements inside cells. To make thermophoresis measurements compatible to cell culture, the setup was changed in great parts, now using total internal reflection fluorescence (TIRF) microscopy.

Actin is one of the most abundant proteins in eukaryotic cells and regulation of the microfilament system is crucial for a wide range of cellular functions including cell shape, cell motility, cell division and membrane dynamics. The aim of this thesis was (1) to gain a better understanding of the function of distinct actin binding domains in the regulation of the actin cytoskeleton and (2) to elucidate the role of actin variants. WH2 domains (WH2, Wiskott-Aldrich syndrome protein homology 2) are ubiquitous multifunctional regulators of actin dynamics. The protein Spire contains four central WH2 domains A-B-C-D with about 20 amino acids each and the cyclase-associated protein CAP2 contains only one WH2 domain. Under certain conditions, they can (1) nucleate actin polymerization, (2) disintegrate actin filaments and (3) sequester actin monomers. Here, the influence of selected Drosophila melanogaster Spire-WH2 and Mus musculus CAP2-WH2 domain constructs on actin dynamics was tested in vitro. To act as a filament nucleator, at least two WH2 domains are required, and nucleation of actin polymerization was only observed at substoichiometric concentrations of WH2 domains over actin. At higher concentrations, the sequestering activity of WH2 domains takes over. Preformed and purified SpireWH2-actin complexes act as extremely efficient nuclei for actin polymerization, even at superstoichiometric WH2 concentrations, under which free WH2 domains would sequester actin. All analyzed constructs, including these with only a single WH2 domain, sequester actin as well as they can disrupt filaments. This latter and most peculiar behavior of WH2 domains was observed in fluorometric, viscometric and TIRF assays. The WH2 domains seem to have such a high affinity for actin that they can forcefully sequester monomers even from filaments and filament bundles, thus breaking the whole structures. Taken together, the data clearly show that SpireWH2-actin complexes are the intermediates that account for the observed nucleating activity, whereas free WH2 domains can disrupt filaments and filament bundles within seconds, again underlining the intrinsic versatility of this regulator of actin dynamics. These data have been confirmed by crystallography in collaboration with the groups of Prof. Dr. Tad Holak and Prof. Dr. Robert Huber (Martinsried, Germany). Besides the well-studied conventional actins many organisms harbor actin variants with unknown function. The model organism Dictyostelium discoideum comprises an actinome of a total of 41 actins, actin isoforms and actin-related proteins. Among them is filactin, a highly conserved actin with an elongated N-terminus. The 105 kDa protein has a distinct domain organization and homologs of this protein are present in other Dictyosteliidae and in some pathogenic Entamoebae. Here, the functions of filactin were studied in vivo and in vitro. Immunofluorescence studies in D. discoideum localize endogenous and GFP-filactin in the cytoplasm at vesicle-like structures and in cortical regions of the cell. A most peculiar behavior is the stress-induced appearance of full length filactin in nuclear actin rods. To perform in vitro analyses recombinant filactin was expressed in Sf9 cells. Fluorescence studies with the filactin actin domain suggest that it interferes with actin polymerization by sequestering G-actin or even capping filaments. Gel filtration assays propose a tetrameric structure of full length filactin. Protein interaction studies suggest that filactin is involved in the ESCRT (endosomal sorting complexes required for transport) pathway which is responsible for multivesicular body formation. The data on filactin suggest that only the conventional actins are the backbone for the microfilamentous system whereas less related actin isoforms have highly specific and perhaps cytoskeleton-independent subcellular functions.

In this work, techniques were developed and used to study the properties of molecules on a single-molecule level. Single-molecule techniques have the major advantage, that in contrast to ensemble measurements, they allow a detailed insight on the distribution and dynamics of single molecules without averaging over subpopulations. The use of Total Internal Reflection Fluorescence Microscopy (TIRFM) in combination with single-pair Förster Resonance Energy Transfer (spFRET) and Alternating Laser Excitation (ALEX) allows the identification of molecular-states by making quantitative measurements of distances in the Ångström range. The development of highly sensitive photon detectors and the use of versatile labeling techniques with photostable (synthetic or genetically-encoded) fluorophores, extended the application of TIRF microscopy to in vitro and live-cell experiments. Despite reducing the complexity of biological systems down to the single-molecule level, functions of individual molecules and interactions between them can be very sophisticated and challenging to analyze. Using information theory based methods, e.g. HMM, the dynamics extracted from single-molecule data was used to illuminate protein interactions and functions. The highly regulated process of gene transcription plays a central role in living organisms. The TATA-box Binding Protein (TBP) is a Transcription Factor (TF) that mediates the formation of the Pre-Initiation Complex (PIC). The lifetime of TBP at the promoter site is controlled by the Modulator of transcription 1 (Mot1), an essential TBP-associated ATPase involved in repression and in activation of transcription. Based on ensemble measurements, various models for the mechanism of Mot1 have been proposed. However, little is known about how Mot1 liberates TBP from DNA. Using TIRF microscopy, the conformation and interaction of Mot1 with the TBP/DNA complex were monitored by spFRET. In contrast to the current understanding of how Mot1 works, Mot1 bound to the TBP/DNA complex is not able to directly disrupt the TBP/DNA complexes by ATP hydrolysis. Instead, Mot1’s ATPase activity induces a conformational change in the complex. The nature of this changed, "primed", conformation is the change of the bending dynamics of the DNA. The results presented in this work suggest a model in which this primed conformation is a destabilized TBP/DNA complex. The interaction with an additional Mot1 molecule is required in order to liberate TBP from DNA. The effect of Mot1 on the DNA dynamics is TBP binding orientation specific. Mot1 effects on the DNA bending dynamics are strongest for molecules where TBP is bound in the inverted binding orientation. The specificity of Mot1’s regulation of DNA bending dynamics suggests that Mot1 preferably "primes" TBP bound in the inverted binding orientation. The mechanistic insight into the interaction of Mot1 with the TBP/DNA complex serves as a framework for understanding the role of Mot1 in gene up- and down-regulation. In a second project, the same single-molecule techniques were used to fabricate and evaluate self-assembled optically controllable, nanodevices. Based on the specificity of Watson-Crick base pairing, DNA was used as a scaffold to position different fluorophores with nanometer accuracy. The functionality of these nanodevices was expanded by making them optically addressable by incorporation of the switchable fluorescent protein Dronpa. Two functions have been demonstrated: Signal enhancement using Optical Lock-In Detection (OLID) and pH sensing in a live-cell environment.

The INO80 complex is a chromatin remodeler involved in diverse nuclear processes like transcriptional regulation, replication fork progression, checkpoint regulation and DNA double strand break repair. In the yeast S. cerevisiae the complex consists of 15 subunits with a total molecular mass of about 1.2 MDa. Knowledge about the atomic structure and molecular architecture of the entire complex is scarce. Similarly, an understanding for the roles of the individual subunits of the complex is mostly lacking. Especially the function of actin and the actin related proteins Arp4 and Arp8 which are found as monomeric components of INO80 and other chromatin remodelers is poorly understood. The goal of this study was to elucidate the functional architecture of the INO80 complex by using a hybrid methods approach. Different structural techniques such as X-ray crystallography, small angle X-ray scattering (SAXS) and electron microscopy (EM) were combined to achieve this goal. Additionally, various functional assays to study the biochemical properties of the actin related proteins and their interaction with actin were employed. In a set of primary experiments expression and purification protocols for seven individual INO80 components, namely Arp4, Arp5, Arp8, Ies4, Ies5, Ies6 and Nhp10 could be established. Additionally, four subcomplexes containing more than one protein, namely Rvb1-Rvb2, Nhp10-Ies5, Nhp10-Ies5-Ies3 and Arp5-Ies6 were purified. Thereby two previously unknown interactions between the INO80 subunits Nhp10 and Ies5, as well as Arp5 and Ies6 could be identified. Subsequently, the newly identified complexes of Nhp10-Ies5-Ies3 and Arp5-Ies6 were studied with SAXS to obtain low resolution solution structures of both. On top of that the entire INO80 complex was purified endogenously from S. cerevisiae and studied by EM. Unfortunately, a three dimensional reconstruction of the remodeler could not be created. Crystallization attempts on all purified INO80 components were successful for the complex of Rvb1-Rvb2 and the actin related protein Arp4. Whereas the structure of Rvb1-Rvb2 could not be solved due to limited diffraction an atomic structure of ATP bound Arp4 at 3.4 Å resolution was obtained. Remarkably, Arp4 does not form filaments despite its high similarity to conventional actin. The lack of polymerization is confirmed by the SAXS structure of isolated Arp4 which indicates it to be monomeric and can be nicely explained on the basis of the crystal structure. Several loop insertions and deletions at positions which are crucial for contact formation within the actin filament, especially at the pointed end of the molecule, prevent Arp4 to engage in filament like interactions. Furthermore, the crystal structure of Arp4 reveals an ATP molecule to be constitutively bound to the protein. The lack of ATPase activity of Arp4 in contrast to actin can be explained with the help of the crystal structure as well. Several residues in the nucleotide clamping loops of Arp4 are divergent from actin leading to a tighter closure and better shielding of the phosphate moieties of the bound ATP from the environment. Most interestingly, Arp4 dramatically influences actin polymerization kinetics. Different fluorescence assays and in vitro TIRF microscopy were used to show that Arp4 is able to inhibit actin polymerization and to depolymerize actin filaments most likely by complex formation with monomeric ADP-actin via the barbed end. Its ability to inhibit actin filament nucleation without sequestering actin while still allowing ADP to ATP exchange within actin resembles the actin binding protein profilin. Arp8 was confirmed by SAXS measurements to be monomeric as well. It is able to sequester actin monomers and to slowly depolymerize actin filaments. Consistent with the formation of a discrete Arp4-Arp8-actin complex within the INO80 remodeler the effects of Arp4 on actin polymerization are further stimulated by Arp8. As both proteins reciprocally enhance their individual effects on actin it is likely that they help to maintain actin in a defined monomeric state within the INO80 chromatin remodeler. The data further suggest a possible assembly between actin and Arp4 via their barbed ends and a model how the Arp4-Arp8-actin complex is integrated into the INO80 chromatin remodeler. Taken together, the findings represent a remarkable advancement in the understanding of nuclear actin related proteins and nuclear actin biochemistry in general. Most excitingly, they indicate a link between chromatin remodeling and nuclear actin dynamics possibly giving chromatin remodeling complexes a role in the actin mediated large scale movement of chromatin.