ABSTRACT
Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow transmission electron microscopy methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with antiphospho-myosin regulatory light chain (MRLC) antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Cytoskeleton/physiology , Myosin Type II/metabolism , Pseudopodia/physiology , Sea Urchins/physiology , Actin-Related Protein 2-3 Complex/ultrastructure , Actins/drug effects , Animals , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Diacetyl/analogs & derivatives , Diacetyl/metabolism , Diacetyl/pharmacology , Enzyme Inhibitors/pharmacology , Microscopy, Electron, Transmission , Myosin Type II/drug effects , Pseudopodia/drug effects , Pseudopodia/ultrastructure , Sea Urchins/drug effectsABSTRACT
The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized by means of the centriole of the fertilizing sperm. In this study, we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5, and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral; displayed metaphase and anaphase-like stages; labeled for centrosomal material, NuMA, and the three mitotic kinesins; and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.
