Dr. Anna Akhmanova (website)
ESR11 York-Christoph Ammon
In polarized epithelial cells, a significant proportion of microtubules is oriented with their plus ends towards the basolateral surface. Recent studies in our lab have shown that an important mechanism of basolateral cortical microtubule attachment relies on the microtubule plus-end tracking proteins EB1, CLASP1/2, MACF2/ACF7 as well as the cortical factors LL5β, ELKS, liprin-α1, β1, and KANK1. The aim of this project is to determine how this network connects to the major polarity complexes, such as Scribble, how it functions in 3D-cultured cells, how it is modified during epithelial-mesenchymal transition and how it is affected by microtubule–targeting agents used in cancer therapy.
- Quantitative description of the role of microtubule-attachment complexes in cortical polarization in 3D breast epithelia acini and their modification during epithelial-mesenchymal transition and invasive growth in 3D.
- Characterization of the biochemical connection between microtubule attachment complexes and the Scribble complex.
- Quantitative characterization of the role of microtubule dynamics in polarity maintenance in 3D-cultured epithelial cells and the effects of microtubule-targeting agents on this process.
Summary of Results
The first part of our project focused on understanding the role of microtubules (MTs) in cell migration under conditions that resemble the physiological conditions better than the common 2D culture systems. For these studies, we used different cancer cell lines grown in collagen I-based soft 3D matrices. To analyze the mechanistic role of MTs in 3D cell migration, we removed different MT plus-end tracking proteins (+TIPs). These +TIPs can have different effects on MTs, some can stabilize MTs whereas others promote MT growth. We were able to demonstrate that 3D cell migration depends on persistent microtubule growth, with the two +TIPs SLAIN2 and CLASP1 being the key factors in this process by suppressing the microtubule catastrophes (the disassembly of MTs) at the cell cortex and promoting cell elongation.
For the second part of our project, we focused on characterizing the KN-motif and ankyrin-repeat-domain containing (KANK) protein family and their role in the cross-talk between microtubules and cell-matrix adhesions. For these studies, we used primarily HeLa cells cultured in 2D. Through pulldown assays in combination with mass spectrometry analysis, we could show that KANK1 binds to the cell-matrix adhesion protein talin. Moreover, since KANK1 can also bind to liprin-b1 and KIF21A, both proteins are part of the cortical microtubule stabilization complex (CMSC) which attaches and stabilizes MTs at the cortex in close proximity to cell-matrix adhesion complexes. On a cellular level, we could demonstrate, by removing endogenous KANKs from HeLa cells via siRNA-mediated knockdown and rescues with different truncated KANK constructs, that the talin-KANK1 interaction is necessary for CMSC organization at the cortex and that the disruption of this interaction disrupts the CMSCs which leads to MT overgrowth at the cell edge.
Another interesting finding about the KANK family proteins is that the different truncated KANK1 and KANK2 constructs show different localization at cell-matrix adhesions. KANK1 and KANK2 constructs that only contain the KN-motif, which mediates binding to talin, completely overlap with the adhesion complexes, whereas full length KANK proteins encircle focal adhesions. So far, we have been able to show that the coiled coil domains of KANK1 and KANK2, which mediate binding to liprins at the cell cortex, and also the linker region of KANK1 and 2, which is located between the coiled coil and the ankyrin-repeat domains of the KANK family proteins, is able to prevent KANK proteins from accumulating inside focal adhesions but rather direct them to focal adhesion periphery. The exact mechanism of how the intrinsically disordered linker region can keep KANK family proteins out of focal adhesions is currently the subject of further research.
- Bouchet, B.P., I. Noordstra, M. van Amersfoort, E.A. Katrukha, Y.C. Ammon, N.D. Ter Hoeve, L. Hodgson, M. Dogterom, P.W. Derksen, and A. Akhmanova. 2016b. Mesenchymal Cell Invasion Requires Cooperative Regulation of Persistent Microtubule Growth by SLAIN2 and CLASP1. Dev Cell. 39:708-723.
- Bouchet, B.P., R.E. Gough, Y.C. Ammon, D. van de Willige, H. Post, G. Jacquemet, A.M. Altelaar, A.J. Heck, B.T. Goult, and A. Akhmanova. 2016a. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions. Elife. 5, e18124.