Cellular Logistics: Unraveling the Interplay Between Microtubule Organization and Intracellular Transport.

Microtubules are core components of the cytoskeleton and serve as tracks for motor protein-based intracellular transport. Microtubule networks are highly diverse across different cell types and are believed to adapt to cell type-specific transport demands. Here we review how the spatial organization of different subsets of microtubules into higher-order networks determines the traffic rules for motor-based transport in different animal cell types. We describe the interplay between microtubule network organization and motor-based transport within epithelial cells, oocytes, neurons, cilia, and the spindle apparatus.

GelMap: intrinsic calibration and deformation mapping for expansion microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.

Oncogene-like induction of cellular invasion from centrosome amplification.

Centrosome amplification has long been recognized as a feature of human tumours; however, its role in tumorigenesis remains unclear. Centrosome amplification is poorly tolerated by non-transformed cells and, in the absence of selection, extra centrosomes are spontaneously lost. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumours, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumour progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behaviour is similar to that induced by overexpression of the breast cancer oncogene ERBB2 (ref. 4) and indeed enhances invasiveness triggered by ERBB2. Our data indicate that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation.

Fibrocystin Is Essential to Cellular Control of Adhesion and Epithelial Morphogenesis.

Mutations of the Pkhd1 gene cause autosomal recessive polycystic kidney disease (ARPKD). Pkhd1 encodes fibrocystin/polyductin (FPC), a ciliary type I membrane protein of largely unknown function, suggested to affect adhesion signaling of cells. Contributions of epithelial cell adhesion and contractility to the disease process are elusive. Here, we link loss of FPC to defective epithelial morphogenesis in 3D cell culture and altered cell contact formation. We study Pkhd1-silenced Madin-Darby Canine Kidney II (MDCKII) cells using an epithelial morphogenesis assay based on micropatterned glass coverslips. The assay allows analysis of cell adhesion, polarity and lumen formation of epithelial spheroids. Pkhd1 silencing critically affects the initial phase of the morphogenesis assay, leading to a reduction of correctly polarized spheroids by two thirds. Defects are characterized by altered cell adhesion and centrosome positioning of FPC-deficient cells in their 1-/2-cell stages. When myosin II inhibitor is applied to reduce cellular tension during the critical early phase of the assay, Pkhd1 silencing no longer inhibits formation of correctly polarized epithelia. We propose that altered sensing and cell interaction of FPC-deficient epithelial cells promote progressive epithelial defects in ARPKD.

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle.

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

Chromatin organizer SATB1 is an important determinant of T-cell differentiation.

T-cell development and differentiation is coordinated by a multitude of signaling molecules and transcription factors that impart distinct functional properties to progenitors. In this review, we focus on the role of the T lineage-enriched chromatin organizer and regulator SATB1 in T-cell differentiation. SATB1 mediates Wnt signaling by recruiting ß-catenin to its genomic targets and coordinates T helper type 2 (T(H)2) differentiation by positively regulating GATA-3. In contrast, maintenance of regulatory T cell (Treg) functions are dependent on inhibition of SATB1-mediated modulation of global chromatin organization. We discuss how regulation of the activity of SATB1 has a critical role in driving these two important differentiation pathways in T cells.

SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation.

Special AT-rich binding protein 1 (SATB1) is a global chromatin organizer and a transcription factor regulated by interleukin-4 (IL-4) during the early T helper 2 (Th2) cell differentiation. Here we show that SATB1 controls multiple IL-4 target genes involved in human Th cell polarization or function. Among the genes regulated by SATB1 is that encoding the cytokine IL-5, which is predominantly produced by Th2 cells and plays a key role in the development of eosinophilia in asthma. We demonstrate that, during the early Th2 cell differentiation, IL-5 expression is repressed through direct binding of SATB1 to the IL-5 promoter. Furthermore, SATB1 knockdown-induced up-regulation of IL-5 is partly counteracted by down-regulating GATA3 expression using RNAi in polarizing Th2 cells. Our results suggest that a competitive mechanism involving SATB1 and GATA3 regulates IL-5 transcription, and provide new mechanistic insights into the stringent regulation of IL-5 expression during human Th2 cell differentiation.

Local changes in microtubule network mobility instruct neuronal polarization and axon specification.

The polarization of neurons into axons and dendrites depends on extracellular cues, intracellular signaling, cytoskeletal rearrangements, and polarized transport, but the interplay between these processes during polarization remains unresolved. Here, we show that axon specification is determined by differences in microtubule network mobility between neurites, regulated by Rho guanosine triphosphatases (GTPases) and extracellular cues. In developing neurons, retrograde microtubule flow prevents the entry of the axon-selective motor protein Kinesin-1 into most neurites. Using inducible assays to control microtubule network flow, we demonstrate that local inhibition of microtubule mobility is sufficient to guide Kinesin-1 into a specific neurite, whereas long-term global inhibition induces the formation of multiple axons. We furthermore show that extracellular mechanical cues and intracellular Rho GTPase signaling control the local differences in microtubule network flow. These results reveal a novel cytoskeletal mechanism for neuronal polarization.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization.

Neuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1-4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

Wnt Signaling Directs Neuronal Polarity and Axonal Growth.

The establishment of neuronal polarity is driven by cytoskeletal remodeling that stabilizes and promotes the growth of a single axon from one of the multiple neurites. The importance of the local microtubule stabilization in this process has been revealed however, the external signals initiating the cytoskeletal rearrangements are not completely understood. In this study, we show that local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth. We found that in the early stages of neuronal polarization, Wnt3a accumulates in one of the neurites of unpolarized cells and thereby could determine axon positioning. Subsequently, Wnt3a localizes to the growing axon, where it activates the canonical Wnt pathway and controls axon positioning and axonal length. We propose a model in which Wnt3a regulates the formation and growth of the axon by activating local intracellular signaling events leading to microtubule remodeling.

Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts.

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell-cell interactions.

Stem Cell-Like Properties of CK2ß-down Regulated Mammary Cells.

The ubiquitous protein kinase CK2 has been demonstrated to be overexpressed in a number of human tumours. This enzyme is composed of two catalytic α or α' subunits and a dimer of ß regulatory subunits whose expression levels are probably implicated in CK2 regulation. Several recent papers reported that unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition, a process involved in cancer invasion and metastasis. Herein, through transcriptomic and miRNA analysis together with comparison of cellular properties between wild type and CK2ß-knock-down MCF10A cells, we show that down-regulation of CK2ß subunit in mammary epithelial cells induces the acquisition of stem cell-like properties associated with perturbed polarity, CD44high/CD24low antigenic phenotype and the ability to grow under anchorage-independent conditions. These data demonstrate that a CK2ß level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Thus, this regulatory subunit functions as a nodal protein to maintain an epithelial phenotype and its depletion drives breast cell stemness.

Polarity Reversal by Centrosome Repositioning Primes Cell Scattering during Epithelial-to-Mesenchymal Transition.

During epithelial-to-mesenchymal transition (EMT), cells lining the tissue periphery break up their cohesion to migrate within the tissue. This dramatic reorganization involves a poorly characterized reorientation of the apicobasal polarity of static epithelial cells into the front-rear polarity of migrating mesenchymal cells. To investigate the spatial coordination of intracellular reorganization with morphological changes, we monitored centrosome positioning during EMT in vivo, in developing mouse embryos and mammary gland, and in vitro, in cultured 3D cell aggregates and micropatterned cell doublets. In all conditions, centrosomes moved from their off-centered position next to intercellular junctions toward extracellular matrix adhesions on the opposite side of the nucleus, resulting in an effective internal polarity reversal. This move appeared to be supported by controlled microtubule network disassembly. Sequential release of cell confinement using dynamic micropatterns, and modulation of microtubule dynamics, confirmed that centrosome repositioning was responsible for further cell disengagement and scattering.

Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells.

Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates.

Spatial segregation between cell-cell and cell-matrix adhesions.

Cell-cell adhesion (CCA) and cell-matrix adhesion (CMA) play determinant roles in the architecture and function of epithelial cells. CCA and CMA are supported by transmembrane molecular complexes that dynamically interact with the extracellular environment and the cell cytoskeleton. Although those complexes have distinct functions, they are involved in a continuous crosstalk. In epithelia, CCA and CMA segregate in distinct regions of the cell surface and thereby take part in cell polarity. Recent results have shown that the two adhesion systems exert negative feedback on each other and appear to regulate actin network dynamics and mechanical force production in different ways. In light of this, we argue that the interplay between these regulatory mechanisms plays an important role in the spatial separation of cell-cell and cell-matrix adhesions components in distinct regions of the cell surface.