Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors

Real-time visualization of clustering and intracellular transport of gold nanoparticles by correlative imaging.

Mechanistic understanding of the endocytosis and intracellular trafficking of nanoparticles is essential for designing smart theranostic carriers. Physico-chemical properties, including size, clustering and surface chemistry of nanoparticles regulate their cellular uptake and transport. Significantly, even single nanoparticles could cluster intracellularly, yet their clustering state and subsequent trafficking are not well understood. Here, we used DNA-decorated gold (fPlas-gold) nanoparticles as a dually emissive fluorescent and plasmonic probe to examine their clustering states and intracellular transport. Evidence from correlative fluorescence and plasmonic imaging shows that endocytosis of fPlas-gold follows multiple pathways. In the early stages of endocytosis, fPlas-gold nanoparticles appear mostly as single particles and they cluster during the vesicular transport and maturation. The speed of encapsulated fPlas-gold transport was critically dependent on the size of clusters but not on the types of organelle such as endosomes and lysosomes. Our results provide key strategies for engineering theranostic nanocarriers for efficient health management

Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as γ-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest

Toward a morphodynamic model of the cell: Signal processing for cell modeling

From a systems biology perspective, the cell is the principal element of information integration. Therefore, understanding the cell in its spatiotemporal context is the key to unraveling many of the still unknown mechanisms of life and disease. This article reviews image processing aspects relevant to the quantification of cell morphology and dynamics. We cover both acquisition (hardware) and analysis (software) related issues, in a multiscale fashion, from the detection of cellular components to the description of the entire cell in relation to its extracellular environment. We then describe ongoing efforts to integrate all this vast and diverse information along with data about the biomechanics of the cell to create a credible model of cell morphology and behavior.Carlos Ortiz-de-Solorzano and Arrate Muñoz-Barrutia were supported by the Spanish Ministry of Economy and Competitiveness grants with reference DPI2012-38090-C03-02 and TEC2013-48552-C02, respectively. Michal Kozubek was supported by the Czech Science Foundation (302/12/G157)

Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis.

The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD

Human Immunodeficiency Virus type 1 Endocytic Trafficking Through Macrophage Bridging Conduits Facilitates Spread of Infection

Bridging conduits (BC) sustain communication and homeostasis between distant tethered cells. These are also exploited commonly for direct cell-to-cell transfer of microbial agents. Conduits efficiently spread infection, effectively, at speeds faster than fluid phase exchange while shielding the microbe against otherwise effective humoral immunity. Our laboratory has sought to uncover the mechanism(s) for these events for human immunodeficiency virus type one (HIV-1) infection. Indeed, in our prior works HIV-1 Env and Gag antigen and fluorescent virus tracking were shown sequestered into endoplasmic reticulum-Golgi organelles but the outcomes for spreading viral infection remained poorly defined. Herein, we show that HIV-1 specifically traffics through endocytic compartments contained within BC and directing such macrophage-to-macrophage viral transfers. Following clathrin-dependent viral entry, HIV-1 constituents bypass degradation by differential sorting from early to Rab11+ recycling endosomes and multivesicular bodies. Virus-containing endocytic viral cargoes propelled by myosin II through BC spread to neighboring uninfected cells. Disruption of endosomal motility with cytochalasin D, nocodasole and blebbistatin diminish intercellular viral spread. These data lead us to propose that HIV-1 hijacks macrophage endocytic and cytoskeletal machineries for high-speed cell-to-cell spread