Project description:Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.
Project description:The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. The first step in DSB repair by homologous recombination is the processing of the ends by one of two resection pathways, executed by the Saccharomyces cerevisiae Exo1 and Sgs1-Dna2 machineries. Here we report in vitro and in vivo studies that characterize the impact of chromatin on each resection pathway. We find that efficient resection by the Sgs1-Dna2-dependent machinery requires a nucleosome-free gap adjacent to the DSB. Resection by Exo1 is blocked by nucleosomes, and processing activity can be partially restored by removal of the H2A-H2B dimers. Our study also supports a role for the dynamic incorporation of the H2A.Z histone variant in Exo1 processing, and it further suggests that the two resection pathways require distinct chromatin remodeling events to navigate chromatin structure.
Project description:Calmodulin (CaM) plays a key role in synaptic function and plasticity due to its ability to mediate Ca(2+) signaling. Therefore, it is essential to understand the dynamics of CaM at dendritic spines. In this study we have explored CaM dynamics using live-cell confocal microscopy and fluorescence recovery after photobleaching (FRAP) to study CaM diffusion. We find that only a small fraction of CaM in dendritic spines is immobile. Furthermore, the diffusion rate of CaM was regulated by neurogranin (Ng), a CaM-binding protein enriched at dendritic spines. Interestingly, Ng did not influence the immobile fraction of CaM at recovery plateau. We have previously shown that Ng enhances synaptic strength in a CaM-dependent manner. Taken together, these data indicate that Ng-mediated enhancement of synaptic strength is due to its ability to target, rather than sequester, CaM within dendritic spines.
Project description:Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression-downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.
Project description:Invadopodia are membrane protrusions dynamically assembled by invasive cancer cells in contact with the extracellular matrix (ECM). Invadopodia are enriched by the structural proteins actin and cortactin as well as metalloproteases such as MT1-MMP, whose function is to degrade the surrounding ECM. During metastasis, invadopodia are necessary for cancer cell intravasation and extravasation. Although signaling pathways involved in the assembly and function of invadopodia are well studied, few studies address invadopodia dynamics and how the cell-ECM interactions contribute to cell invasion. Using iterative analysis based on time-lapse microscopy and mathematical modeling of invasive cancer cells, we found that cells oscillate between invadopodia presence and cell stasis-termed the "invadopodia state"-and invadopodia absence during cell translocation-termed the "migration state." Our data suggest that ?1-integrin-ECM binding and ECM cross-linking control the duration of each of the two states. By changing the concentration of cross-linkers in two-dimensional and three-dimensional cultures, we generate an ECM in which 0-0.92 of total lysine residues are cross-linked. Using an ECM with a range of cross-linking degrees, we demonstrate that the dynamics of invadopodia-related functions have a biphasic relationship to ECM cross-linking. At intermediate levels of ECM cross-linking (0.39), cells exhibit rapid invadopodia protrusion-retraction cycles and rapid calcium spikes, which lead to more frequent MT1-MMP delivery, causing maximal invadopodia-mediated ECM degradation. In contrast, both extremely high or low levels of cross-linking lead to slower invadopodia-related dynamics and lower ECM degradation. Additionally, ?1-integrin inhibition modifies the dynamics of invadopodia-related functions as well as the length of time cells spend in either of the states. Collectively, these data suggest that ?1-integrin-ECM binding nonlinearly translates small physical differences in the extracellular environment to differences in the dynamics of cancer cell behaviors. Understanding the conditions under which invadopodia can be reduced by subtle environment-targeting treatments may lead to combination therapies for preventing metastatic spread.
Project description:Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.
Project description:We have identified Rab10 as an ER-specific Rab GTPase that regulates ER structure and dynamics. We show that Rab10 localizes to the ER and to dynamic ER-associated structures that track along microtubules and mark the position of new ER tubule growth. Rab10 depletion or expression of a Rab10 GDP-locked mutant alters ER morphology, resulting in fewer ER tubules. We demonstrate that this defect is due to a reduced ability of dynamic ER tubules to grow out and successfully fuse with adjacent ER. Consistent with this function, Rab10 partitions to dynamic ER-associated domains found at the leading edge of almost half of all dynamic ER tubules. Interestingly, this Rab10 domain is highly enriched with at least two ER enzymes that regulate phospholipid synthesis, phosphatidylinositol synthase (PIS) and CEPT1. Both the formation and function of this Rab10/PIS/CEPT1 dynamic domain are inhibited by expression of a GDP-locked Rab10 mutant. Together, these data demonstrate that Rab10 regulates ER dynamics and further suggest that these dynamics could be coupled to phospholipid synthesis.
Project description:Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.
Project description:Human telomeres possess a single-stranded DNA (ssDNA) overhang of TTAGGG repeats, which can self-fold into a G-quadruplex structure. POT1 binds specifically to the telomeric overhang and partners with TPP1 to regulate telomere lengthening and capping, although the mechanism remains elusive. Here, we show that POT1 binds stably to folded telomeric G-quadruplex DNA in a sequential manner, one oligonucleotide/oligosaccharide binding fold at a time. POT1 binds from 3' to 5', thereby unfolding the G-quadruplex in a stepwise manner. In contrast, the POT1-TPP1 complex induces a continuous folding and unfolding of the G-quadruplex. We demonstrate that POT1-TPP1 slides back and forth on telomeric DNA and also on a mutant telomeric DNA to which POT1 cannot bind alone. The sliding motion is specific to POT1-TPP1, as POT1 and ssDNA binding protein gp32 cannot recapitulate this activity. Our results reveal fundamental molecular steps and dynamics involved in telomere structure regulation.
Project description:The defining event in apoptosis is mitochondrial outer membrane permeabilization (MOMP), allowing apoptogen release. In contrast, the triggering event in primary necrosis is early opening of the inner membrane mitochondrial permeability transition pore (mPTP), precipitating mitochondrial dysfunction and cessation of ATP synthesis. Bcl-2 proteins Bax and Bak are the principal activators of MOMP and apoptosis. Unexpectedly, we find that deletion of Bax and Bak dramatically reduces necrotic injury during myocardial infarction in vivo. Triple knockout mice lacking Bax/Bak and cyclophilin D, a key regulator of necrosis, fail to show further reduction in infarct size over those deficient in Bax/Bak. Absence of Bax/Bak renders cells resistant to mPTP opening and necrosis, effects confirmed in isolated mitochondria. Reconstitution of these cells or mitochondria with wild-type Bax, or an oligomerization-deficient mutant that cannot support MOMP and apoptosis, restores mPTP opening and necrosis, implicating distinct mechanisms for Bax-regulated necrosis and apoptosis. Both forms of Bax restore mitochondrial fusion in Bax/Bak-null cells, which otherwise exhibit fragmented mitochondria. Cells lacking mitofusin 2 (Mfn2), which exhibit similar fusion defects, are protected to the same extent as Bax/Bak-null cells. Conversely, restoration of fused mitochondria through inhibition of fission potentiates mPTP opening in the absence of Bax/Bak or Mfn2, indicating that the fused state itself is critical. These data demonstrate that Bax-driven fusion lowers the threshold for mPTP opening and necrosis. Thus, Bax and Bak play wider roles in cell death than previously appreciated and may be optimal therapeutic targets for diseases that involve both forms of cell death.