RESUMO
Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.
Assuntos
Leishmania donovani/citologia , Leishmania donovani/metabolismo , Lisossomos/metabolismo , Fagossomos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Dictyostelium/citologia , Dineínas/metabolismo , Glicoesfingolipídeos/metabolismo , Microdomínios da Membrana/metabolismo , CamundongosRESUMO
Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that, using the true pairwise distribution, we can define and maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images.
Assuntos
Algoritmos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Artefatos , Corantes Fluorescentes/química , Processos EstocásticosRESUMO
SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE-DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Transporte Biológico , DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Microscopia Eletrônica , Ligação Proteica , Conformação ProteicaRESUMO
DNA intercalators are widely used in cancer therapeutics, to probe protein-DNA interactions and to investigate the statistical-mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43%, while not affecting its mechanical properties.
Assuntos
DNA/efeitos dos fármacos , Corantes Fluorescentes/química , Mutagênicos/química , DNA/química , Dano ao DNA , Fluorescência , Corantes Fluorescentes/farmacologia , Mutagênicos/farmacologia , Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Raios UltravioletaRESUMO
Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.
Assuntos
Cromatina , Sinais (Psicologia) , Humanos , Histonas/genética , Citoesqueleto , Tecido ConjuntivoRESUMO
Dense matrices impede interstitial cell migration and subsequent repair. We hypothesized that nuclear stiffness is a limiting factor in migration and posited that repair could be expedited by transiently decreasing nuclear stiffness. To test this, we interrogated the interstitial migratory capacity of adult meniscal cells through dense fibrous networks and adult tissue before and after nuclear softening via the application of a histone deacetylase inhibitor, Trichostatin A (TSA) or knockdown of the filamentous nuclear protein Lamin A/C. Our results show that transient softening of the nucleus improves migration through microporous membranes, electrospun fibrous matrices, and tissue sections and that nuclear properties and cell function recover after treatment. We also showed that biomaterial delivery of TSA promoted in vivo cellularization of scaffolds by endogenous cells. By addressing the inherent limitations to repair imposed by nuclear stiffness, this work defines a new strategy to promote the repair of damaged dense connective tissues.
RESUMO
Long-distance transport of many organelles inside eukaryotic cells is driven by the dynein and kinesin motors on microtubule filaments. More than 30 years since the discovery of these motors, unanswered questions include motor- organelle selectivity, structural determinants of processivity, collective behaviour of motors and track selection within the complex cytoskeletal architecture, to name a few. Fluorescence microscopy has been invaluable in addressing some of these questions. Here we present a review of some efforts to understand these sub-microscopic machines using fluorescence.
Assuntos
Dineínas/metabolismo , Células Eucarióticas/metabolismo , Cinesinas/metabolismo , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Miosinas/metabolismo , Animais , Anticorpos/química , Bioensaio , Transporte Biológico , Dineínas/química , Células Eucarióticas/química , Células Eucarióticas/ultraestrutura , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinesinas/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/instrumentação , Microtúbulos/química , Microtúbulos/ultraestrutura , Miosinas/química , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Organelas/química , Organelas/metabolismo , Organelas/ultraestruturaRESUMO
Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients' antibodies cause cross-linking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients' antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs.