Superresolution imaging of HIV in infected cells with FlAsH-PALM

Plexins and semaphorins comprise a large family of receptorligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled β1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual β1 integrin catch bonds. Loss of plexinD1 expression reduces β1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.thymocyte development | mechanobiology | integrin activation | autoimmunity | central tolerance I t is well established that plexins and their semaphorin ligands regulate biological processes in multiple physiological domains including axon pathfinding (1, 2), angiogenesis (3), and immunity (4). Although plexins harbor potential for regulating small GTPases that mediate cytoskeletal remodeling, either through a direct cytoplasmic GTPase-activating protein (GAP) domain and Rac/Rho-GTPase binding domain or through sequestration of guanidine nucleotide exchange factor (GEF) proteins to the membrane-proximal cytoplasmic face, there is little consensus on plexin signaling pathways in a complex physiological context (5).While studying the molecular interactions controlling mouse thymocyte development, we identified plexinD1 as a critical mediator for thymocytes destined to mature into T lymphocytes populating the mammalian immune system (6). Plxnd1 encoding plexinD1 is transcriptionally regulated at a key intermediate stage of thymocyte development. Double-negative (DN) thymocytes lacking expression of CD4 and CD8 as well as plexinD1 differentiate in the thymic cortex into largely nondividing CD4 + CD8 + double-positive (DP) thymocytes that display surface αβ T-cell receptors (TCRs) and express plexinD1 (6). Despite being highly mobile (7), DP thymocytes remain sequestered in the cortex in frequent physical association with thymic epithelial cells (TECs), moving toward the thymic medulla via chemokine guidance only subsequent to TCR stimulation by self-derived peptide/MHC complexes (pMHC) that induce CD69 expression and support cell survival, i.e., positive selection (8, 9). CD69 + DP cells differentiate further into CD4 + CD8 − or CD4 − CD8 + singlepositive (SP) thymocytes, translocating to the thymic medulla to complete their maturation (10, 11). While traversing the thymus, immatu...

Section: Resultsmentioning

confidence: 99%

Dynamic control of β1 integrin adhesion by the plexinD1-sema3E axis

Choi

Duke‐Cohan

Chen

et al. 2013

“…However, GFP-Vpr dissociates from the RTCs/PICs shortly after infection and has not been observed in the nuclei of infected cells (33,36,37). To date, only a small number of studies have detected PICs in the nuclei of infected cells by using virions labeled with fluorescently tagged IN (38)(39)(40)(41), and one recent study showed that there are nuclear IN complexes that are not associated with viral cDNA (42), suggesting that fluorescently tagged IN may not be a suitable marker for labeling PICs in the nuclei. Analysis of the nuclear import of PICs has largely relied on quantification of 2-LTR circles, which requires completion of reverse transcription (43).…”

Section: Discussionmentioning

confidence: 99%

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

Burdick

Pathak

2013

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) are host factors that incorporate into virions and restrict virus replication. We labeled HIV-1 particles with yellow fluorescent protein (YFP)-tagged APOBEC3 proteins and examined their association with preintegration complexes (PICs) in infected cells. Labeling of PICs with A3F-YFP, and to a lesser extent A3G-YFP, could be used to visualize PICs in the nuclei, which was dependent on nuclear pore protein Nup153 but not TNPO3. We show that reverse transcription is not required for nuclear import of PICs, indicating that a viral core uncoating event associated with reverse transcription, and the central DNA flap that forms during reverse transcription, are not required for nuclear import. We also quantify association of cytoplasmic PICs with nuclear envelope (NE) and report that capsid mutations that increase or decrease core stability dramatically reduce NE association and nuclear import of PICs. In addition, we find that nuclear PICs remain close to the NE and are not distributed throughout the nuclei. These results provide tools for tracking retroviral PICs in infected cells and reveal insights into HIV-1 replication.retrovirus | microscopy | early events

“…HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus-host interplay during all steps of HIV-1 replication cycle (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14,15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture.…”

mentioning

confidence: 99%

Single-Cell Imaging of HIV-1 Provirus (SCIP)

Primio

Quercioli

Allouch

et al. 2013

Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.T echnical developments in imaging-based techniques have greatly improved our understanding of HIV-host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus-host interplay during all steps of HIV-1 replication cycle (1-13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14, 15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA (16, 17). Here we describe a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. We exploited a site-specific genome engineering technique that represents one of the most promising approaches to detect specific genome regions in modified organism (18) allowing for their spatial localization in the cell (19,20). This technique couples endogenous repair pathways, induced by rare cutting endonuclease, with immunofluorescence analysis. Rare cutting endonucleases, such as the yeast-homing endonuclease I-SceI, specifically cuts target sequences that cannot be found in the mammalian genome. By engineering DNA to contain the I-SceI cleavage site, it is thus possible to induce endogenous repair mechanism for double-strand breaks (DSBs) at specific genomic positions. DSB repair leads to the formation of distinct subnuclear structures that are generally referred to as "foci" (21). The first sensor of the DSB is the histone H2AX, which becomes massively phosphorylated at serine 139 (γ-H2AX). Foci of DNA repair are thus visible through immu...