An arginine-rich nuclear localization signal (ArgiNLS) strategy for streamlined image segmentation of single-cells.

High-throughput volumetric fluorescent microscopy pipelines can spatially integrate whole-brain structure and function at the foundational level of single-cells. However, conventional fluorescent protein (FP) modifications used to discriminate single-cells possess limited efficacy or are detrimental to cellular health. Here, we introduce a synthetic and non-deleterious nuclear localization signal (NLS) tag strategy, called 'Arginine-rich NLS' (ArgiNLS), that optimizes genetic labeling and downstream image segmentation of single-cells by restricting FP localization near-exclusively in the nucleus through a poly-arginine mechanism. A single N-terminal ArgiNLS tag provides modular nuclear restriction consistently across spectrally separate FP variants. ArgiNLS performance in vivo displays functional conservation across major cortical cell classes, and in response to both local and systemic brain wide AAV administration. Crucially, the high signal-to-noise ratio afforded by ArgiNLS enhances ML-automated segmentation of single-cells due to rapid classifier training and enrichment of labeled cell detection within 2D brain sections or 3D volumetric whole-brain image datasets, derived from both staining-amplified and native signal. This genetic strategy provides a simple and flexible basis for precise image segmentation of genetically labeled single-cells at scale and paired with behavioral procedures.

WIP/ITSN1 complex is involved in cellular vesicle trafficking and formation of filopodia-like protrusions.

WIP (WASP interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) regulates actin polymerization that is crucial for invadopodia and filopodia formation. Recently, we reported the WIP interaction with ITSN1 which is highly implicated in endo-/exocytosis, apoptosis, mitogenic signaling and cytoskeleton rearrangements. Here we demonstrate that the WIP/ITSN1 complex is involved in the transferrin receptor recycling and partially co-localizes with a marker of the fast recycling endosomes, RAB4. Moreover, ITSN1 recruits WIP to RAB4-positive vesicles upon overexpression. Our data indicate that WIP enhances the interaction of N-WASP with ITSN1 and promotes ITSN1/ß-actin association. Moreover, the WIP/ITSN1-L complex facilitates formation of filopodia-like protrusions in MCF-7 cells. Thus, WIP/ITSN1 complex is involved in the cellular vesicle trafficking and actin-dependent membrane processes.

F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis.

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin "patches" form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

Correlated light and electron microscopy of cell division in large marine oocytes, eggs, and embryos.

The rapid and synchronous divisions of large and transparent oocytes, eggs, and embryos of marine species are exceptionally well suited for microscopic observation. Consequently, these cells have been models for cell division research since its beginnings and contributed some of its first and most fundamental discoveries. While large size and rapid transitions render these cells ideal specimens for light microscopy, the same features constitute a challenge for electron microscopy. Here, we describe example protocols from our work on starfish oocyte meiosis, where we overcome these challenges by using live imaging of fluorescently labeled structures in combination with correlated electron microscopy. In this work, we demonstrate how: (i) to capture a rapid, transient event in time and (ii) to localize a small structure within the large oocyte. These techniques are applicable with minor modifications to oocytes and embryos of other species and, possibly, to other cell types.

Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia.

Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.