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Biomedical subjects

Eric Karsenti

Publications and source records attributed to Eric Karsenti.

At least 19 recordsLinked to original sources
PubMed2007

Discrete states of a protein interaction network govern interphase and mitotic microtubule dynamics.

The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.

Animals↗
PubMed2006

Gradients in the self-organization of the mitotic spindle.

Recent evidence points at a role of protein interaction gradients around chromatin in mitotic spindle morphogenesis in large eukaryotic cells. Here, we explain how gradients can arise over distances of tens of microns around supramolecular structures from mixtures of soluble molecules. We discuss how coupled sets of such reaction diffusion processes generate the spatial information that determines the local dynamics of microtubules required to form a bipolar spindle. We argue that such reaction diffusion processes are involved in the self-organization of supramolecular structures in the cell.

Animals↗
PubMed2006

Modelling microtubule patterns.

The cellular cytoskeleton is well studied in terms of its biological and physical properties, making it an attractive subject for systems approaches. Here, we describe the experimental and theoretical strategies used to study the collective behaviour of microtubules and motors. We illustrate how this led to the beginning of an understanding of dynamic cellular patterns that have precise functions.

Adenosine Triphosphate↗
PubMed2006

Detection and quantification of protein-microtubules interactions using green fluorescent protein photoconversion.

We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be detected and binding kinetics can be determined rapidly and accurately. This method provides an alternative to classical in vitro microtubule-binding assays to analyze microtubule-associated proteins binding to microtubules. It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes.

Green Fluorescent Proteins↗
PubMed2005

Microtubule-dependent transport and organization of sarcomeric myosin during skeletal muscle differentiation.

It has been proposed that microtubules (MTs) participate in skeletal muscle cell differentiation. However, it is still unclear how this happens. To examine whether MTs could participate directly in the organization of thick and thin filaments into sarcomeres, we observed the concomitant reorganization and dynamics of MTs with the behavior of sarcomeric actin and myosin by time-lapse confocal microscopy. Using green fluorescent protein (GFP)-EB1 protein to label MT plus ends, we determined that MTs become organized into antiparallel arrays along fusing myotubes. Their dynamics and orientation was found to be different across the thickness of the myotubes. We observed fast movements of Dsred-myosin along GFP-MTs. Comparison of GFP-EB1 and Dsred-myosin dynamics revealed that myosin moved toward MT plus ends. Immuno-electron microscopy experiments confirmed that myosin was actually associated with MTs in myotubes. Finally, we confirmed that MTs were required for the stabilization of myosin-containing elements prior to incorporation into mature sarcomeres. Collectively, our results strongly suggest that MTs become organized into a scaffold that provides directional cues for the positioning and organization of myosin filaments during sarcomere formation.

Actins↗
PubMed2005

Spatial coordination of spindle assembly by chromosome-mediated signaling gradients.

During cell division, chromosomes are distributed to daughter cells by the mitotic spindle. This system requires spatial cues to reproducibly self-organize. We report that such cues are provided by chromosome-mediated interaction gradients between the small guanosine triphosphatase (GTPase) Ran and importin-beta. This produces activity gradients that determine the spatial distribution of microtubule nucleation and stabilization around chromosomes and that are essential for the self-organization of microtubules into a bipolar spindle.

Animals↗
PubMed2005

TPX or not TPX?

An Aurora A regulatory module has been identified in two different proteins: TPX2 in Xenopus laevis and TPXL-1 in C. elegans. The diverse roles of these two proteins in spindle assembly leave us to beckon the true C. elegans TPX2 ortholog to center stage.

Animals↗
PubMed2004

Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked.

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.

Animals↗
PubMed2004

Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts.

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

Animals↗
PubMed2004

Stathmin-tubulin interaction gradients in motile and mitotic cells.

The spatial organization of the microtubule cytoskeleton is thought to be directed by steady-state activity gradients of diffusible regulatory molecules. We visualized such intracellular gradients by monitoring the interaction between tubulin and a regulator of microtubule dynamics, stathmin, using a fluorescence resonance energy transfer (FRET) biosensor. These gradients were observed both during interphase in motile membrane protrusions and during mitosis around chromosomes, which suggests that a similar mechanism may contribute to the creation of polarized microtubule structures. These interaction patterns are likely to reflect phosphorylation of stathmin in these areas.

Animals↗
PubMed2004

Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome.

The application of functional proteomics to important model organisms with unsequenced genomes is restricted because of the limited ability to identify proteins by conventional mass spectrometry (MS) methods. Here we applied MS and sequence-similarity database searching strategies to characterize the Xenopus laevis microtubule-associated proteome. We identified over 40 unique, and many novel, microtubule-bound proteins, as well as two macromolecular protein complexes involved in protein translation. This finding was corroborated by electron microscopy showing the presence of ribosomes on spindles assembled from frog egg extracts. Taken together, these results suggest that protein translation occurs on the spindle during meiosis in the Xenopus oocyte. These findings were made possible due to the application of sequence-similarity methods, which extended mass spectrometric protein identification capabilities by 2-fold compared to conventional methods.

Animals↗
PubMed2004

The mitotic spindle and actin tails.

To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.

Actins↗
PubMed2003

Long-range communication between chromatin and microtubules in Xenopus egg extracts.

The mitotic spindle of animal cells is a bipolar array of microtubules that guides chromosome segregation during cell division. It has been proposed that during spindle assembly chromatin can positively influence microtubule stability at a distance from its surface throughout its neighboring cytoplasm. However, such an "à distance" effect has never been visualized directly. Here, we have used centrosomal microtubules and chromatin beads to probe the regulation of microtubule behavior around chromatin in Xenopus egg extracts. We show that, in this system, chromatin does affect microtubule formation at a distance, inducing preferential orientation of centrosomal microtubules in its direction. Moreover, this asymmetric distribution of microtubules is translated into a directional migration of centrosomal asters toward chromatin and their steady-state repositioning within 10 microm of chromatin. To our knowledge, this is the first direct evidence of a long-range guidance effect at the sub-cellular level.

Animals↗
PubMed2003

Importin alpha-regulated nucleation of microtubules by TPX2.

The importin alpha-regulated microtubule-associated protein TPX2 is known to be critical for meiotic and mitotic spindle formation in vertebrates, but its detailed mechanism of action and regulation is not understood. Here, the site of interaction on TPX2 for importin alpha is mapped. A TPX2 mutant that cannot bind importin alpha is constitutively active in the induction of microtubule-containing aster-like structures in Xenopus egg extract, demonstrating that no other importin alpha or RanGTPase target is required to mediate microtubule assembly in this system. Further, recombinant TPX2 is shown to induce the formation and bundling of microtubules in dilute solutions of pure tubulin. In this purified system, importin alpha prevents TPX2-induced microtubule formation, but not TPX2-tubulin interaction or microtubule bundling. This demonstrates that TPX2 has more than one mode of interaction with tubulin and that only one of these types of interaction is abolished by importin alpha. The data suggest that the critical early function in spindle formation regulated by importin alpha is TPX2-mediated microtubule nucleation.

Animals↗
PubMed2003

Stu2p and XMAP215: turncoat microtubule-associated proteins?

The dynamics of microtubule-based (MT) cytoskeletons are controlled by a variety of accessory proteins: microtubule-associated proteins (MAPs), which usually stabilize MTs, and microtubule-destabilizers. Two related MAPs, XMAP215 and Stu2p, are known to stabilize MTs. However, recent studies report that these proteins have a MT-destabilizing function as well. Here we discuss the implications of these reports.

Animals↗
PubMed2003

Self-organisation and forces in the microtubule cytoskeleton.

Modern microscopy techniques allow us to observe specifically tagged proteins in live cells. We can now see directly that many cellular structures, for example mitotic spindles, are in fact dynamic assemblies. Their apparent stability results from out-of-equilibrium stochastic interactions at the molecular level. Recent studies have shown that the spindles can form even after centrosomes are destroyed, and that they can even form around DNA-coated beads devoid of kinetochores. Moreover, conditions have been produced in which microtubule asters interact even in the absence of chromatin. Together, these observations suggest that the spindle can be experimentally deconstructed, and that its defining characteristics can be studied in a simplified context, in the absence of the full division machinery.

Animals↗
PubMed2002

Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein.

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.

Actins↗