Biology subjects

van Engelenburg, S. B.

Publications and source records attributed to van Engelenburg, S. B..

4 recordsLinked to original sources

bioRxiv2026

ER shaping proteins guide spindle elongation and division during rapid cleavage mitoses

The ER is a complex network of membranes that inhabits much of the cytoplasm of cells - however, this network undergoes a massive condensation and rapid remodeling during cell division. In Drosophila cleavage divisions, this results in a tight association of the ER with centrosomes and mitotic spindle poles. Previous work has shown that this relationship between the ER and centrosomes must be finely tuned to enable successful spindle elongation, and that overaccumulation of the ER in these stages can result in failed centrosome maturation. During interphase, the ER exists in tubular and sheet-like arrangements, with a variety of "shaping" proteins enforcing these topologies. Here, we examine the contributions of these ER shaping proteins to the rapid changes that occur during cleavage mitoses in the Drosophila embryo. A screen of ER shaping proteins revealed that disruption of Reep-family proteins leads to mitotic failures at characteristic cleavage stages. Compromising ReepA, the Drosophila ortholog of the Reep1-4 subfamily, had a lesser impact on early embryonic mitoses. However, ReepB (the ortholog of the Reep5-6 subfamily) disruption, significantly affects ER mitotic coat morphologies, resulting in a frilled ER phenotype and a reduction of ER adherence to the spindle space accompanied by division failures. Overexpressing ReepA does not rescue ReepB mitotic or ER morphology defects and instead introduces local condensates of abnormal ER structures. These data suggest that dedicated Reep proteins guide ER mitotic properties at specific early developmental stages. Using a cell-based in vitro analysis of Drosophila Reeps, we identify differential "tubulating" properties of ReepA and ReepB. Together these data suggest that the minutes-scale ER remodeling required for early mitoses is governed by shaping proteins, and that ReepB family members are especially important in some of the most rapid cleavage divisions that occur in early embryo.

cell biology↗

bioRxiv2022

Rab11-FIP1C is dispensable for HIV-1 replication in primary CD4+ T cells but its role is cell-type-dependent in immortalized human T-cell lines

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that siRNA-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR/Cas9 technology was used to knock out the expression of this protein in several human T-cell lines - Jurkat E6.1, SupT1 and H9 - and in primary human CD4+ T cells. FIP1C knock-out caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T-cell lines. In H9 cells, FIP1C knock-out caused a cell-density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knock-out had no effect on HIV-1 replication. Furthermore, HTLV-I transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail - Y795W - implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. ImportanceThe incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. Gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types - human T-cell lines and primary CD4+ T cells - we used CRISPR/Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

microbiology↗

bioRxiv2020

Isotropic 3D electron microscopy reference library of whole cells and tissues

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) demonstrated the ability to image cellular samples at 4-nm isotropic voxels with rather limited imageable volume. Here, we present 3D EM images of whole cells and tissues with two orders of magnitude increases in imageable volume at 4-nm voxels. Such data with a combined fine resolution scale and large sample size do not currently exist, and are enabled by the advances in higher precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning. More importantly, we have generated a volume EM atlas encompassing ten diverse datasets of whole cells and tissues, from cancer cells to immune cells, and from mouse pancreatic islets to Drosophila neural tissues. These open-access data (via OpenOrganelle) represent a foundation to nucleate a new field of high-resolution whole-cell volume EM and subsequent analyses, and invite biologists to explore this new paradigm and pose fundamentally new questions.

cell biology↗

bioRxiv2020

Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail

HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study we investigated the viral and cellular factors that contribute to the permissivity of MT-4 to gp41 CT truncation. We found that the kinetics of HIV-1 production are faster in MT-4 than in the other T-cell lines tested, but MT-4 express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 express higher levels of plasma-membrane-associated Env than non-permissive cells and Env internalization from the plasma membrane is slower compared to another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4, two-fold less Env is incorporated into virus particles in MT-4 compared to SupT1. Cell-to-cell transmission between co-cultured 293T and MT-4 is higher than in co-cultures of 293T with most other T-cell lines tested, indicating that MT-4 are highly susceptible to this mode of infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.Importance The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 transmission. This study reveals that multiple factors contribute to the unique ability of a gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a requirement for the gp41 CT in MT-4 is associated with the combined effects of rapid HIV-1 protein production, high levels of cell-surface Env expression, and increased susceptibility to cell-to-cell transmission compared to non-permissive cells.List of abbreviationsCTcytoplasmic tailVSvirological synapseC-Ccell-to-cellEnvenvelope glycoproteinSTRshort tandem repeatMFImedian fluorescence intensityPMplasma membraneRadio-IPradio-immunoprecipitationhPBMChuman peripheral blood mononuclear cellsRTreverse transcriptaseVSV-GVesicular stomatitis virus glycoproteinPBSphosphate buffered salineFBSfetal bovine serumWTwild typeHRPhorseradish peroxidaseERendoplasmic reticulumMAHIV-1 Matrix proteinEBVEpstein-Barr virusHTLVHuman T-cell leukemia virusALLAcute lymphocytic leukemiaATLAdult T-cell lymphomaATCCAmerican Type Culture CollectionNIH ARPNIH AIDS Reagent Programn.s.not statistically significantINHIV-1 Integrase proteinCAHIV-1 Capsid proteinRINRNA Integrity NumberView Full Text

microbiology↗