Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the ...centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion.
The microtubule cytoskeleton regulates cell polarity by spatially organizing membrane trafficking and signaling processes. In epithelial cells, microtubules form parallel arrays aligned along the ...apico-basal axis, and recent work has demonstrated that the members of CAMSAP/Patronin family control apical tethering of microtubule minus ends. Here, we show that in mammalian intestinal epithelial cells, the spectraplakin ACF7 (also known as MACF1) specifically binds to CAMSAP3 and is required for the apical localization of CAMSAP3-decorated microtubule minus ends. Loss of ACF7 but not of CAMSAP3 or its homolog CAMSAP2 affected the formation of polarized epithelial cysts in three-dimensional cultures. In short-term epithelial polarization assays, knockout of CAMSAP3, but not of CAMSAP2, caused microtubule re-organization into a more radial centrosomal array, redistribution of Rab11-positive (also known as Rab11A) endosomes from the apical cell surface to the pericentrosomal region and inhibition of actin brush border formation at the apical side of the cell. We conclude that ACF7 is an important regulator of apico-basal polarity in mammalian intestinal cells and that a radial centrosome-centered microtubule organization can act as an inhibitor of epithelial polarity.
Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family ...members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.
The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that ...CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes.
•CAMSAP2 stabilizes microtubule minus ends at the Golgi•A complex of AKAP450 and myomegalin recruits CAMSAP2-bound microtubules to the Golgi•A single Golgi can be maintained in the absence of centrosome and Golgi microtubules•Golgi microtubules facilitate cell reorientation and migration in 3D matrix
Wu et al. describe how a microtubule minus-end-binding protein, CAMSAP2, acts together with Golgi-associated proteins AKAP450 and myomegalin to promote microtubule organization at the Golgi. They also show how these proteins affect cell migration and describe the redundancy between the centrosome-dependent and -independent pathways in microtubule minus-end stabilization.
The motor protein kinesin-1 plays an important role in polarized sorting of transport vesicles to the axon. However, the mechanism by which the axonal entry of kinesin-1-dependent cargo transport is ...regulated remains unclear. Microtubule-associated protein MAP7 (ensconsin in Drosophila) is an essential kinesin-1 cofactor and promotes kinesin-1 recruitment to microtubules. Here, we found that MAP7 family member MAP7D2 concentrates at the proximal axon, where it overlaps with the axon initial segment and interacts with kinesin-1. Depletion of MAP7D2 results in reduced axonal cargo entry and defects in axon development and neuronal migration. We propose a model in which MAP7D2 in the proximal axon locally promotes kinesin-1-mediated cargo entry into the axon.
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•The microtubule-binding domain of MAP7D2 targets the proximal axon•MAP7D2 is required for axon development•MAP7D2 associates with all three kinesin-1 family members•MAP7D2 promotes kinesin-1-mediated cargo transport in axons
Kinesin-1 is well known for transporting vesicles and organelles into to the axon. Pan et al. show that MAP7D2 concentrates at the proximal axon, regulates kinesin-1 activity, and locally promotes kinesin-1-mediated cargo trafficking into axons. Specific microtubule-associated proteins (MAPs) that locally control motor activities is an emerging concept.
Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on ...the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.
Microtubule organization has a crucial role in regulating cell architecture. The geometry of microtubule arrays strongly depends on the distribution of sites responsible for microtubule nucleation ...and minus-end attachment. In cycling animal cells, the centrosome often represents a dominant microtubule-organizing center (MTOC). However, even in cells with a radial microtubule system, many microtubules are not anchored at the centrosome, but are instead linked to the Golgi apparatus or other structures. Non-centrosomal microtubules predominate in many types of differentiated cell and in mitotic spindles. In this review, we discuss recent advances in understanding how the organization of centrosomal and non-centrosomal microtubule networks is controlled by proteins involved in microtubule nucleation and specific factors that recognize free microtubule minus ends and regulate their localization and dynamics.
Microtubule minus-end organization in interphase and mitotic animal cells should be envisioned beyond the centrosome-centered view.
MTOC activity can be separated into a nucleating and an anchoring function, which require some common and distinct factors.
Microtubule minus-end organization in spindles depends on their nucleation, stabilization, and motor-based microtubule sliding.
The MTOC activities of the centrosome and the Golgi share common regulators and compete with each other.
An increasing number of regulatory proteins recognize free, uncapped microtubule minus ends and regulate their dynamics.
The interprotofilament interface is an important site for specific microtubule end recognition.
Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for ...Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish.
We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties.
Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.
Rasa3 is a GTPase activating protein of the GAP1 family which targets R-Ras and Rap1. Although catalytic inactivation or deletion of Rasa3 in mice leads to severe hemorrhages and embryonic lethality, ...the biological function and cellular location of Rasa3 underlying these defects remains unknown. Here, using a combination of loss of function studies in mouse and zebrafish as well as in vitro cell biology approaches, we identify a key role for Rasa3 in endothelial cells and vascular lumen integrity. Specific ablation of Rasa3 in the mouse endothelium, but not in megakaryocytes and platelets, lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice. Reduced plexus/sprouts formation and vascular lumenization defects were observed when Rasa3 was specifically inactivated in mouse endothelial cells at the postnatal or adult stages. Similar results were obtained in zebrafish after decreasing Rasa3 expression. In vitro, depletion of Rasa3 in cultured endothelial cells increased β1 integrin activation and cell adhesion to extracellular matrix components, decreased cell migration and blocked tubulogenesis. During migration, these Rasa3-depleted cells exhibited larger and more mature adhesions resulting from a perturbed dynamics of adhesion assembly and disassembly which significantly increased their life time. These defects were due to a hyperactivation of the Rap1 GTPase and blockade of FAK/Src signaling. Finally, Rasa3-depleted cells showed reduced turnover of VE-cadherin-based adhesions resulting in more stable endothelial cell-cell adhesion and decreased endothelial permeability. Altogether, our results indicate that Rasa3 is a critical regulator of Rap1 in endothelial cells which controls adhesions properties and vascular lumen integrity; its specific endothelial cell inactivation results in occluded blood vessels, hemorrhages and early embryonic death in mouse, mimicking thus the Rasa3-/- mouse phenotype.
Sjögren’s syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in ...SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients
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