Michel Bornens

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.

Centrosome composition and microtubule anchoring mechanisms

Centrosomes of animal cells and spindle pole bodies of fungi are the major microtubule nucleating centers. Recent studies indicate that their capacity to organize microtubule arrays rests on elaborate control of the anchoring and release of the nucleated microtubules. Although common molecular mechanisms are likely to be involved in both cases, the centrosome from animal cells shows considerable complexity and flexibility, which contrasts with the simple laminar organization of spindle pole bodies in fungi. The role of the centriole pair in controlling both the structural stability and the activity of the centrosome in animal cells is now becoming clearer. The potential use of the generational asymmetry of centrosomes or spindle pole bodies for controlling cell polarity is also a growing theme.

Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell–cell or cell–extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus–centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.

Experimental and theoretical study of mitotic spindle orientation

The architecture and adhesiveness of a cell microenvironment is a critical factor for the regulation of spindle orientation in vivo. Using a combination of theory and experiments, we have investigated spindle orientation in HeLa (human) cells. Here we show that spindle orientation can be understood as the result of the action of cortical force generators, which interact with spindle microtubules and are activated by cortical cues. We develop a simple physical description of this spindle mechanics, which allows us to calculate angular profiles of the torque acting on the spindle, as well as the angular distribution of spindle orientations. Our model accounts for the preferred spindle orientation and the shape of the full angular distribution of spindle orientations observed in a large variety of different cellular microenvironment geometries. It also correctly describes asymmetric spindle orientations, which are observed for certain distributions of cortical cues. We conclude that, on the basis of a few simple assumptions, we can provide a quantitative description of the spindle orientation of adherent cells.

Centrosome organization and centriole architecture: their sensitivity to divalent cations.

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved (i) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also observed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A scheme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization.

The Centrosome in Cells and Organisms

Centrosome Center Stage? The centrosome is a major organizer of the cytoskeleton in animal cells. The precise duplication of this organelle at each cell cycle ensures proper organization of the mitotic apparatus and chromosome segregation. However, centrosomes are dispensable for cell division during some stages of development in mouse and Drosophila. Bornens (p. 422) reviews the role of the centrosome in a variety of organisms and discusses how and why they can be dispensed with on occasion. In this vein, Azimzadeh et al. (p. 461, published online 5 January) now demonstrate that centrosomes in Planarian flatworms, best known for their remarkable regeneration abilities, are completely dispensable for both cell division and development. The centrosome acts as the main microtubule-nucleating organelle in animal cells and plays a critical role in mitotic spindle orientation and in genome stability. Yet, despite its central role in cell biology, the centrosome is not present in all multicellular organisms or in all cells of a given organism. The main outcome of centrosome reproduction is the transmission of polarity to daughter cells and, in most animal species, the sperm-donated centrosome defines embryo polarity. Here I will discuss the role of the centrosome in cell polarity, resulting from its ability to position the nucleus at the cell center, and discuss how centrosome innovation might have been critical during metazoan evolution.

External forces control mitotic spindle positioning

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

Microtubule nucleation at the cis-side of the Golgi apparatus requires AKAP450 and GM130

We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal γ‐TuRC‐interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis‐Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin‐A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis‐side of the Golgi in an MT‐independent, GM130‐dependent manner. Short AKAP450‐dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi‐associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome–Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome‐associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.

Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A‐kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21‐22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled‐coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII‐binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA‐buffer extracts of HeLa cells demonstrated co‐precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co‐purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.