An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.

Abstract

Abstract. Rhodamine-labeled monoclonal antibodies, which react with tyrosinated a-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475 were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

Reassessing the Role and Dynamics of Nonmuscle Myosin II during Furrow Formation in EarlyDrosophilaEmbryos

“…That MTs and microfilaments are required for cellularization is well established (Zalokar and Erk, 1976;Foe and Alberts, 1983;Edgar et al, 1987;Warn et al, 1987;Sisson et al, 2000). Treatment with colchicine or cytochalasin at the onset of cellularization completely inhibits furrow ingression just as it inhibits metaphase furrow invagination.…”

“…It is well established from studies of fixed embryos that colchicine or cytochalasin treatment blocks cellularization (Zalokar and Erk, 1976;Foe and Alberts, 1983;Edgar et al, 1987;Warn et al, 1987;Sisson et al, 2000). We took advantage of the RLC-GFP marker to specifically follow the in vivo dynamics of furrow progression and myosin II network reorganization in response to various treatments (Table 2).…”

“…Metaphase furrow formation and cellularization share several properties with conventional cytokinesis: both kinds of invaginations involve actin cytoskeleton rearrangements, and this reorganization depends (directly or indirectly) on microtubules (MTs; Zalokar and Erk, 1976;Foe and Alberts, 1983;Edgar et al, 1987;Warn et al, 1987;Foe et al, 2000). In addition, several other known cortical components of the classical cytokinesis contractile rings are found concentrated within the metaphase furrows and the furrow canals of cellularizing embryos (Zalokar and Erk, 1976;Foe and Alberts, 1983).…”

Targeting antibodies to the cytoplasm

“…In addition, their biochemical properties can be changed at will, e.g., by fusion to other protein domains, or they can even be immediately expressed inside the living cell to induce knockdown phenotypes. 6,7 The usefulness of antibodies in visualization or the knockdown of protein functions in the living cell has been proven by microinjection, [8][9][10][11][12] which demonstrated the stability and function of mature antibodies in the living cytoplasm. However, examples published since the first report of this method are scarce, mainly *Correspondence to: Stefan Dübel; Email: s.duebel@tu-bs.de Submitted: 10/13/10; Accepted: 11/04/10 DOI: 10.4161/mabs.3.…”

Interdependence of Filamentous Actin and Microtubules for Asymmetric Cell Division

“…Yet no direct data is available that interaction of astral microtubules with cortical actin is responsible for the rearrangement into actin caps. It has been shown, however, that injection of antibodies against tubulin, which results in spindle collapse and disappearance of the nuclei from the cortex, also abolished the actin caps (Warn et al, 1987). Further support came from the discovery of conditions that promote movement of syncytial nuclei away from the cortex and induce loss of cortical actin caps/rings (Foe et al, 2000).…”