WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation.

WASP-family proteins are known to promote assembly of branched actin networks by stimulating the filament-nucleating activity of the Arp2/3 complex. Here, we show that WASP-family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP-family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline-rich sequence that binds profilin-actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline-rich sequences are required to support polymerase activity by (i) bringing polymerization-competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin-actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP-family proteins that create it. Collaboration between WH2 and proline-rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP-family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation

Multifunctionality of membrane remodeling proteins Reticulon and Dynamin 2

170 p.Los procesos de remodelación de membranas, especialmente aquellos en los que la transformación supone un proceso irreversible, como son la fusión y la fisión, son mecanismos vitales de las células eucariotas para su correcto funcionamiento, tanto a nivel interno, como entre el medio extracelular y el intracelular.Para poder realizarse tales procesos, las maquinarias moleculares (complejos proteicos) se encargan de interactuar con las membranas diana mediante distintos mecanismos (proteínas citosólicas), o bien forman parte de la propia membrana mediante largos dominios hidrofóbicos actuando como anclas. En ambos casos, tales proteínas tienden a oligomerizar para poder formar complejos funcionales a la hora de generar la energía necesaria para ser capaces de llevar a cabo procesos de fisión y fusión.En esta tesis doctoral, me he centrado en el estudio de dos proteínas encargadas de la remodelación de la membrana, la Dinamina 2 (citosólica) y el Reticulon (transmembrana). En ambos casos, hemos descubierto como las características físico-químicas, así como la geometría de las membranas que serán remodeladas, son de vital importancia para la transición de estas proteínas a complejos funcionalmente activos en la fisión de las membranas. Así pues, el presente trabajo revela como las membranas diana juegan un papel clave en la activación de la actividad remodeladora de complejos proteicos específico

Systems analysis of early endosome motility through identification of molecular motors

Endocytosis is an evolutionary conserved process of internalization of cargo from the extracellular environment, be they ligands, nutritional and signaling or pathogens into cells. Following their entry, cargo is received into vesiculo-tubular network of early endosomal compartments from where they are sorted and routed to appropriate cellular destinations through transport along the endocytic network. Recycling cargo is sorted away from other cargo resident in early endosomes through tubulation resulting in fission of recycling vesicles, while those to be degraded are progressively concentrated in early endosomes to be degraded in lysosomes. Early endosomes are dynamic organelles that have been shown to move centripetally following the internalization of cargo into at the cell periphery. Their motility from the cell periphery to the juxtanuclear location of the cell involves convoluted trajectories that include directed motility, bi-directional switches, saltatory behavior and stalls. This complex motility presumably contributes toward the cargo sorting, duration of cargo residence and spatio-temporal signaling by early endosomes. How the different regimes of motility, and nature and number of molecular motors involved in early endosome motility contribute toward endosome function is not understood. The aim of this study was to probe into the regulation of endosome motility and understand how transport organizes early endosome network. Towards this end, live cell time-lapse movies of Rab5 endosomes were analyzed to derive motility properties contributing to organization of early endosomes. Consistent and significant bias toward the cell centre (minus end motility) in kinetic parameters such as speed, displacement and duration of motility contribute to centripetal flux of Rab5 early endosomes. A phenomenological property of early endosome motility is its saltatory behavior that produces saturation curves in Mean Square Displacement (MSD) plots. This phase of motility is descriptive, with no understanding of its mechanism or function. Live cell candidate RNAi screen and cytoskeletal perturbation analysis were performed to identify molecules regulating saltatory motility. To this end, cellular microtubule perturbation and RNAi knock down of several Kinesin motor candidates showed a loss in saturation behavior. Potential candidates identified have to be tested for their effect on endosome function through cargo sorting and kinetic assays to gain insights into the role of saltatory motility in endosome function. Molecular motors mediate Rab5 motility. Therefore, understanding regulation of motility requires identifying number and nature of molecular motors involved in their transport. Towards this end, a functional cargo (LDL) degradation RNAi screen targeting molecular motors was performed. The Ambion Select technology was used with 3 siRNAs targeting every gene in the library. Analysis of screen produced by lack of phenotype consistency between the multiple siRNAs targeting the same gene. Hence, a search for technology with better target specificity was initiated. Technologies tested were Ambion Select, Ambion Silencer Select, Dharmacon ON-TARGET Plus, esiRNA and Invitrogen Stealth. Invitrogen Stealth technology was found to produce the least off-targets and was most specific in terms of consistency of phenotypes produced by multiple siRNAs silencing the same target gene. Assay conditions were also found to influence the silencing specificities to a significant extent. Hence, a systematic assay optimization exercise was performed in terms of the concentration of siRNA used for transfection and time window of assay to maximize specificity of siRNA silencing. Insights obtained from methodologies developed herein not only provide invaluable guidelines in choosing RNAi commercial libraries for screens, but also underscore the importance of establishing optimal assay conditions to minimize off-targets and improve specificity of silencing target genes. The motor screen was repeated with RNAi library from Invitrogen Stealth. Several potentially interesting candidates have been identified. Also, correlation analyses of phenotypes produced in the screen have indicated toward potential regulatory motor complexes, all of which await biochemical validation

Nucleoporin mRNA localization and Annulate Lamellae biosynthesis during Drosophila melanogaster oogenesis

Nuclear pore complexes (NPCs) are large protein assemblies that connect the eukaryotic nucleus with the cytoplasm, thus facilitating all transport between them. Besides the nuclear envelope (NE), NPCs also occur in parallel stacks of cytoplasmic membranes called Annulate Lamellae (AL) that can serve as storage, facilitating rapid nuclear growth via NE insertion during fruit fly embryogenesis. How and when AL are assembled is largely unknown. In this work, I established that AL are already abundant in late stage oocytes, and progressively accumulate throughout oogenesis specifically in the oocyte. By screening the localization of 39 nucleoporin and related mRNAs, I detected the specific enrichment of two nucleoporin and three importin encoding transcripts to AL, the NE, and previously unidentified nucleoporin granules throughout the egg chamber. Perturbation experiments revealed a dependence on active translation, but independence of an intact microtubule network on mRNA localization. Generation of GFP::Nup358 transgenic flies revealed granules with distinct partial nucleoporin contents, that are subject to microtubule-dependent transport and interactions among them. Their spatiotemporal abundance distribution is indicative of NPC precursors, and they contain partial accumulations of pore complexes within internal membranes. These granules further displayed characteristics of biomolecular condensates, including fast intra-granule dynamics, exclusion of cytoplasmic constituents, and sensitivity to 1,6-hexanediol. Both condensation state and AL assembly were dependent on Ran, a protein also fundamental for NPC assembly at the NE. Its nucleotide status throughout this is likely controlled by differential localization of its modulators RanGAP and Rcc1 to granules and cytoplasm respectively. This work thus established a molecular framework and basic sequence of events that leads to the assembly of AL, which are crucial during early development, and might have broader implications for NPC assembly also at the NE

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in <i>Arabidopsis</i>

Background&lt;p&gt;&lt;/p&gt; The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell’s endomembrane, cytoskeletal and signal transduction apparatuses.&lt;p&gt;&lt;/p&gt; Results&lt;p&gt;&lt;/p&gt; In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic “unmasking” of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling.&lt;p&gt;&lt;/p&gt; Conclusions&lt;p&gt;&lt;/p&gt; Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism

Beyond transcription : a post-transcriptional role of 3D chromatin crosstalk in oncogene regulation

This thesis explores how stochastic chromatin fibre interactions, chromatin organization in the 3D nuclear architecture, and environmental signals collaborate to regulate MYC oncogene expression in human colon cancer cells. In Paper I, we employ the ultra-sensitive Nodewalk technique to uncover the dynamic and stochastic nature of chromatin networks impinging on MYC. The analyses revealed that the MYC interactome mainly consists of stochastic pairwise interactions between MYC and its flanking enhancers in two neighbouring topologically associated domains (TADs), which are insulated self-interacting genomic domains. The limits of Nodewalk were also pushed to enable the detection of interactions in very small cell populations, corresponding to the genomic content of ~7 cells. Comparing the frequency of interactions detected in such small input samples with ensemble interactomes of large cell populations uncovered that the enhancer hubs of the ensemble interactomes that appear to simultaneously interact with MYC likely represent virtual events, which are not present in reality at the single cell level. These data support a model where MYC interacts with its enhancers in a mutually exclusive way, with MYC screening for enhancer contacts, rather than the other way around. Paper II provides a detailed understanding of a novel post-transcriptional mechanism of enhancer action on MYC expression. We have thus uncovered that the cancer-specific recruitment of the MYC gene to nuclear pores and ensuing rapid nuclear export of MYC transcripts - a process that increases MYC expression by enabling the escape of MYC mRNAs from rapid decay in the nucleus - require a CTCF binding site positioned within the colorectal oncogenic super-enhancer. Genetic editing by CRISPR-Cas9 was thus commissioned to establish two clones of human colon cancer cells with a mutated sequence in the OSE-specific CTCFBS. Comparing the mutant cells to the parental cell line, we uncovered that the WNT-dependent increase in the nuclear export rate of MYC transcripts was abrogated in the CTCFBS mutant clones, providing the first genetic evidence of super- enhancer-mediated gene gating in human cells. In line with this finding, the OSE-specific CTCFBS thus conferred a significant growth advantage to the parental colon cancer cells, compared to the mutant clones. Moreover, we found that WNT-dependent CCAT1 eRNA transcription is mediated by the OSE-specific CTCFBS that is required for recruitment of AHCTF1 to the OSE to mediate the positioning of the OSE to the nuclear periphery, enabling the subsequent facilitation of MYC mRNA export. A multistep molecular process including WNT signalling and the OSE-specific CTCFBS thus underlies the gene gating of MYC in human colon cancer cells, and could potentially be targeted for diagnostic or therapeutic uses. In summary, this thesis explores the dynamics of the stochastic interactomes impinging on the MYC oncogene, and provides new insights on the role of 3D chromatin orchestration in the transcriptional regulation of MYC. Our analyses uncovered the molecular factors involved in the gene gating of MYC, and thus increase our understanding of tumour development. These findings could potentially be beneficial for future diagnostic approaches, or for targeted therapeutic strategies in the treatment of cancer

Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network

Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells

Developing a Cell-like Substrate to Investigate the Mechanosensitivity of Cell-to-Cell Junctions

Indiana University-Purdue University Indianapolis (IUPUI)The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties