Open Biol. 2017 Feb;7(2). pii: 160274.

Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to noncentrosomal microtubule organizing centres


Deborah A. Goldspink1, Chris Rookyard2, Benjamin J. Tyrrell1, Jonathan Gadsby1, James Perkins1, Elizabeth K. Lund1, Niels Galjart4, Paul Thomas1, Tom Wileman3 and Mette M. Mogensen1

1School of Biological Sciences, 2School of Computing Science, and 3Medical School, University of East Anglia, Norwich, UK and 4Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands



Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how MTOCs are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apicobasal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.

Open Biol. 7: 160274.



Main findings

Microtubules are vital for many cellular processes including intracellular transport and cell division, polarity and differentiation. How microtubules are organised is critical and needs to reflect cell function. Control of microtubule organisation is the responsibility of the Microtubule Organising Centre (MTOC) that acts as a hub regulating their temporal and spatial organisation. A radial microtubule array emanating from a centrally located centrosomal MTOC is prominent in many animal cells. Here the microtubule tracks can transport material to and from the cell periphery and this is well suited for relatively flat cells. In contrast, columnar epithelial cells, such as those of the kidney and intestine, assemble non-radial transcellular arrays that allow selective transport of material to the top and bottom of the cell and thus better support the specialised functions of these cells. New MTOCs (known as non-centrosomal MTOCs (n-MTOCs)) that are distinct from the centrosome assembly are part of this process (1-3). Formation of n-MTOCs is therefore critical as they are responsible for anchorage and organisation of the transcellular microtubules and thus influence epithelial cell polarity, shape and function, with defects in polarity leading to loss of tissue architecture and function. However, how n-MTOCs form is not fully understood.

Here we show using a combination of in vitro epithelial cell and organoid cultures and ex-vivo intestinal tissue that the microtubule anchoring protein ninein is required for transcellular microtubule arrays formation and that the microtubule plus-end tracking protein CLIP-170 is needed for its relocation to n-MTOCs. CLIP-170 knockdown in cultured cells or knockout in mouse intestinal cells resulted in absence of ninein at the n-MTOC without causing a decrease in ninein at the centrosome or in overall expression (Figure 1). The findings also revealed roles for IQGAP1 and active Rac1 in ninein relocalisation and a model was proposed in which IQGAP1 acts as a receptor for the capture of CLIP-170 tipped microtubules in a process promoted by active Rac1 thus enabling ninein translocation to n-MTOCs. CLIP-170 may alternatively, or in addition, act as a receptor and together with IQGAP1 and active Rac1 form a complex for the capture of ninein at the n-MTOC.



Figure 1. Intestinal tissue reveal lack of ninein at apical n-MTOCs in Clip1/Clip2 double knockout. Confocal images of villus cells labeled for ninein (red) and b-catenin (green) and stained for DNA with DAPI (blue) showing columnar cells with ninein concentrated at the apical n-MTOCs in wildtype but mainly cytoplasmic/centrosomal ninein in the knockout. Scale bar= 5 μm. Open Biology 7: 160272


Interestingly, although ninein is critical for anchorage of microtubule minus-ends at the centrosome (4) it is not essential for microtubule anchorage at the n-MTOC. In fact, lack of ninein at the n-MTOC does not prevent formation of apico-basal microtubule arrays as long as ninein is expressed (Figure 2). This suggests that a compensation mechanism is operating to ensure microtubule minus-end anchorage at the n-MTOC. Indeed, the centrosomal anchoring protein p150Glued and microtubule minus-end stabiliser CAMSAP2 were present at n-MTOCs in both wild-type and knockout mouse intestinal tissue and organoids. However, other studies have shown that loss of CAMSAP3 compromises microtubule organisation although overall apico-basal polarity is maintained (5-6). It is therefore likely that a complex of proteins is responsible for microtubule minus-end anchorage at n-MTOCs and that loss of one or more of these components may produce varying degrees of microtubule-anchorage defects.



Figure 2. Apico-basal microtubules are evident in organoids generated from both wild-type and Clip1/Clip2 double knockout intestinal stem cells. Confocal images of cells from villus domains in organoids labeled for microtubules (blue) and b-catenin (red) showing distinct apico-basal microtubule arrays within columnar cells in both wildtype and knockout. Scale bar = 5 μm. Open Biology 7: 160272




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