Christian Cibert

Initiation of gastrulation in the mouse embryo is preceded by an apparent shift in the orientation of the anterior-posterior axis.

BACKGROUNDnIt is generally assumed that the migration of anterior visceral endoderm (AVE) cells from a distal to a proximal position at embryonic day (E)5.5 breaks the radial symmetry of the mouse embryo, marks anterior, and conditions the formation of the primitive streak on the opposite side at E6.5. Transverse sections of a gastrulating mouse embryo fit within the outline of an ellipse, with the primitive streak positioned at one end of its long axis. How the establishment of anterior-posterior (AP) polarity relates to the morphology of the postimplantation embryo is, however, unclear.nnnRESULTSnTransverse sections of prestreak E6.0 embryos also reveal an elliptical outline, but the AP axis, defined by molecular markers, tends to be perpendicular to the long axis of the ellipse. Subsequently, the relative orientations of the AP axis and of the long axis change so that when gastrulation begins, they are closer to being parallel, albeit not exactly aligned. As a result, most embryos briefly lose their bilateral symmetry when the primitive streak starts forming in the epiblast.nnnCONCLUSIONSnThe change in the orientation of the AP axis is only apparent and results from a dramatic remodeling of the whole epiblast, in which cell migrations take no part. These results reveal a level of regulation and plasticity so far unsuspected in the mouse gastrula.

Inhibition of flagellar beat frequency by a new anti-β-tubulin antibody

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Bending of the “9+2” axoneme analyzed by the finite element method

Many data demonstrate that the regulation of the bending polarity of the 9+2 axoneme is supported by the curvature itself, making the internal constraints central in this process, adjusting either the physical characteristics of the machinery or the activity of the enzymes involved in different pathways. Among them, the very integrated Geometric Clutch model founds this regulation on the convenient adjustments of the probability of interaction between the dynein arms and the beta-tubulin monomers of the outer doublet pairs on which they walk. Taking into consideration (i) the deviated bending of the outer doublets pairs (Cibert, C., Heck, J.-V., 2004. Cell Motil. Cytoskeleton 59, 153-168), (ii) the internal tensions of the radial spokes and the tangential links (nexin links, dynein arms), (iii) a theoretical 5 microm long proximal segment of the axoneme and (iv) the short proximal segment of the axoneme, we have reevaluated the adjustments of these intervals using a finite element approach. The movements we have calculated within the axonemal cylinder are consistent with the basic hypothesis that found the Geometric Clutch model, except that the axonemal side where the dynein arms are active increases the intervals between the two neighbor outer doublet pairs. This result allows us to propose a mechanism of bending reversion of the axoneme, involving the concerted ignition of the molecular engines along the two opposite sides of the axoneme delineated by the bending plane.

The angle of the dorsoventral axis with respect to the anteroposterior axis in the Drosophila embryo is controlled by the distribution of gurken mRNA in the oocyte

Like most organisms, Drosophila embryos develop in relation to two orthogonal axes, the anteroposterior and dorsoventral. These two axes are established by four independent systems of maternal information. Mutations in any system disrupt either the anteroposterior or the dorsoventral patterning of the embryo but never affect the orthogonal orientation of the axes relative to each other. Here we show by analyzing their embryonic fate map, that K10 embryos still possess a dorsoventral polarity. However, instead of forming a 90 degrees angle, the dorsoventral and the anteroposterior axes lie parallel to each other. This axis misorientation was partially corrected by decreasing the wild-type grk gene copy number such that the embryos issued from K10/K10; grk/+ females showed a variability in their fate map which could be interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anteroposterior axis. This rotation is maximal in the K10 embryos where it reaches 90 degrees and results in the congruence of the two axes. The alteration of the embryonic fate map could be traced back to oogenesis where it was shown to correlate with the mislocalization of the grk transcripts.

Is the curvature of the flagellum involved in the apparent cooperativity of the dynein arms along the ?9+2? axoneme?

In a recent study [Cibert, 2008. Journal of Theoretical Biology 253, 74-89], by assuming that walls of microtubules are involved in cyclic compression/dilation equilibriums as a consequence of cyclic curvature of the axoneme, it was proposed that local adjustments of spatial frequencies of both dynein arms and beta-tubulin monomers facing series create propagation of joint probability waves of interaction (JPI) between these two necessary partners. Modeling the occurrence of these probable interactions along the entire length of an axoneme between each outer doublet pair (without programming any cooperative dialog between molecular complexes) and the cyclic attachment of two facing partners, we show that such constituted active couples are clustered. Along a cluster the dynein arms exhibit a small phase shift with respect to the order according to which they began their cycle after being linked to a beta-tubulin monomer. The number of couples included in these clusters depends on the probability of interaction between the dynein arms and the beta-tubulin, on the location of the outer doublet pairs around the axonemal cylinder, and on the local bending of the axoneme; around the axonemal cylinder, the faster and the larger the sliding, the shorter the clusters. This mechanism could be involved in the apparent cooperativity of molecular motors and the beta-tubulin monomers, since it is partially controlled by local curvature, and the cluster length is inversely proportional to the sliding activity of the outer doublet pairs they link.

Elastic axonemal model: Solitary wave shapes

We introduce an elastic beam Bernoulli-Euler model for the axoneme (cytoskeletal inner core of eukaryotic cells appendages) whose dynamics is controlled by internally generated torques at the inflexion points of the beam. We calculate the geometrical and the mechanical parameters of the axoneme, and compared them with experimental data. We make predictions about nonlinear shapes, including possible solitary waves in the curvature, which can explain certain swimming patterns of the cells.

Real-time observation of microtubules attached to microtubule organizing centers in vitro*

Summary— The dynamics and organization of microtubules associated with axonemes and kinetochores in vitro were visualized using video microscopy techniques. Microtubules attached either at the ends of axonemes or to mitotic chromosomes behave accordining to dynamic instability in our conditions. Microtubules attached to kinetochores showed lower rates of elongation and shortening than those nucleated by axonemes in the same conditions. In addition, elementary bundles of microtubules appeared spontaneously in association with kinetochores, with microtubules elongating along previously attached microtubules at even lower rates. Such side interactions, either spontaneous or stabilized by factors such as MAPs, might affect microtubule dynamics directly.