SnapShot: Nucleo-cytoskeletal Interactions.

The nucleus is connected to the cytoskeleton, and these connections are involved in multiple functions such as nuclear positioning, shape and stiffness, cytoskeleton organization, mechanotransduction, gene expression, chromosome positioning, DNA repair, and cell migration.

Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

BACKGROUND: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood. RESULTS: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling. CONCLUSIONS: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration.

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4-7 Different cell types position their nuclei away from the leading edge during cell migration.8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15-22 and the actin regulator Eps8L223-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

Methods to Measure Perinuclear Actin Dynamics During Nuclear Movement in Migrating Cells.

The nucleus is specifically positioned within a cell in diverse biological contexts. There are multiple connections between the nuclear envelope and the cytoskeleton and these connections are involved in nuclear positioning. During cell polarization prior to cell migration, nuclear envelope proteins bind to the actin cytoskeleton and get organized into linear arrays, known as transmembrane actin-associated nuclear (TAN) lines to move the nucleus away from the leading edge. Here we describe methods to study perinuclear actin dynamics, including measurement of the thickness of actin cables coupled to TAN lines, measurement of the number of perinuclear actin cables, and ablation of perinuclear actin cables. These methods are used to identify mechanisms of nuclear positioning.

Nuf and Rip11 requirement for polarity determinant recycling during Drosophila development.

A tight relationship between apico-basal polarity and trafficking is essential for epithelial physiology and tissue homeostasis. Recent studies have described how some Rab GTPases, key components of the intracellular traffic machinery, contribute to the establishment of cell polarity in vertebrates. We have demonstrated a novel connection between cell polarity and trafficking: in Drosophila epithelia, the apical determinant aPKC is recycled via Rab11-Nuf-recycling endosomes to maintain cell polarity. Furthermore, the phosphorylation of Nuf by aPKC allows aPKC to control the sub-cellular localization of Nuf and its own membrane accumulation. Here we review these data and show the different contribution of the 2 Drosophila Rab11 adaptor proteins, Nuf and Rip11, to the maintenance of Drosophila embryonic ectoderm polarity.

Dealing with the nucleus during cell migration.

The position of the nucleus within cells is a key event during cell migration. The movement and positioning of the nucleus strongly impacts cell migration. Notably, the last two years largely contributed to emphasise the dynamicity of the nucleus-cytoskeleton interactions that occur during cell migration. Nuclei are under continuous tension from opposing intracellular forces and its tether to the cytoskeleton can be regulated at different levels. Interestingly, it was showed how nuclear positioning is highly related to cell function. In most migrating cells, including cancer cells, the nucleus can be the rate limiting step of cell migration and is placed away from the leading edge. By contrast, leukocytes position their nucleus close to the lamellipodia at the leading edge, and the nucleus contributes to drilling through the endothelium. Differences in cell migration in 2D versus 3D environments are also evident. The mechanisms and forces at play during nuclear positioning and translocation are clearly affected by the nature of the substrate. As such nuclear positioning during cell migration can vary between cell types and environments. In this review we aim to give an overview of the latest discoveries in the field revealing how nuclear positioning is tightly regulated, not only by intrinsic nuclear properties, such as deformability, nuclear envelope content or nucleus-cytoskeleton connectivity, but also by the microenvironment.