David R. Burgess

Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis

Cytokinesis in animal cells is accomplished in part by an actomyosin contractile ring. Recent work on amphibian, Drosophila, and Caenorhabditis elegans embryos implicates membrane trafficking and delivery as essential for cytokinesis. However, the relative contributions of contractile ring constriction versus membrane insertion to cytokinesis and the temporal relationship between these processes are largely unexplored. Here we monitor secretion of the extracellular matrix protein, hyalin, as a marker for new plasma membrane addition in dividing sea urchin zygotes. We find that new membrane addition occurs specifically in the cleavage furrow late in telophase independent of contractile ring constriction. The directed equatorial deposition of new furrow membrane requires astral microtubules and release of internal stores of Ca2+, but not the presence of a central spindle. Further, cells arrested in M phase do not secrete hyalin, suggesting that mitotic exit is required for new membrane addition. These results demonstrate that astral overlap in equilaterally dividing cells not only serves to specify positioning and contraction of the contractile ring, but also to direct the delivery of new membrane to the furrow as a late, independent event during cytokinesis.

The cytoskeleton in development of epithelial cell polarity

SUMMARY The polarization of intestinal epithelial cells and the stereotypic arrangement of their actin-based cytoskeleton have made these epithelia an excellent system to explore the organization and formation of a cortical actin-based cytoskeleton. Through a combined morphological and biochemical analysis, the molecular arrangement of many of the components of the brush border has been elucidated. Study of brush border assembly in the Crypts of Lieberkühn suggests that cytoskeletal mRNA and protein expression, as well as morphological development, occur rapidly following cell differentiation. Protein kinases appear to be important regulators of intestinal cell growth, for differentiating cells in the crypts possess 15-fold higher levels of tyrosine phosphorylated proteins than differentiated cells of the villus. One of these kinases, pp60c-src, has a 4- to 7-fold higher activity in crypts and increased association with the cytoskeleton than it has in villus cells. The development and maintenance of polarization in epithelial cells require the targeting and transport of specific proteins to the apical and basolateral plasma membrane. It has been proposed that a dynein-like, microtubule-based motor is involved in the transport of apically directed materials from the trans-Golgi to the apical plasma membrane. However, microtubules do not reach the plasma membrane, but terminate below the actin-rich network of filaments comprising the terminal web. We propose that vesicles translocate from the Golgi to the apical cytoplasm along microtubules using dynein, and then move through the terminal web to reach the apical plasma membrane using the actin-based motor myosin-I. Our isolation of Golgi-derived vesicles possessing both myosin-I and dynein on their cytoplasmic surface is consistent with this hypothesis.

Budding roles for myosin II on the Golgi.

Myosin II--conventional myosin--has been typecast in muscle-man roles. While members of the Schwarzenegger clan from skeletal muscle have grabbed the limelight, myosin II motors in nonmuscle cells labour away in many varied and subtle roles. Recent findings show that nonmuscle myosin II, along with other myosins and cytoskeletal proteins, assembles on Golgi membranes. Nonmuscle myosin II associates transiently with membranes of the trans-Golgi network during the budding of a subpopulation of transport vesicles. The exact role of myosin II in vesicular trafficking is not yet understood, but its participation heralds a novel role for actin-based motors in vesicle budding.

Microvillus Assembly: Not actin alone

Transfection studies provide supporting evidence for the proposed role of villin and fimbrin in bundling the core actin filaments of microvilli.

MEMBRANE MOTILITY MEDIATED BY UNCONVENTIONAL MYOSIN

Largely on the basis of their physical properties and their localization to cell membranes, it has been proposed that the unconventional myosins are membrane motors. In the past year, a combination of immunological, biochemical and genetic approaches has begun to provide direct evidence that unconventional myosins have important roles in movements of the plasma membrane and cytoplasmic organelles.

Interaction between EB1 and p150glued Is Required for Anaphase Astral Microtubule Elongation and Stimulation of Cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.

Transitions Regulating the Timing of Cytokinesis in Embryonic Cells

Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.

‘Life is a Highway’: Membrane Trafficking During Cytokinesis

Cytokinesis, the final stage of the cell cycle, is an essential step toward the formation of two viable daughter cells. In recent years, membrane trafficking has been shown to be important for the completion of cytokinesis. Vesicles originating from both the endocytic and secretory pathways are known to be shuttled to the plasma membrane of the ingressing cleavage furrow, delivering membrane and proteins to this dynamic region. Advances in cell imaging have led to exciting new discoveries regarding vesicle movement in living cells. Recent work has revealed a significant role for membrane trafficking, as controlled by regulatory proteins, during cytokinesis in animal cells. The endocytic and secretory pathways as well as motor proteins are revealed to be essential in the delivery of vesicles to the cleavage furrow during cytokinesis.

Light microscopy of echinoderm embryos.

Publisher Summary The chapter discusses commonly used procedures for visualizing both fixed and live echinoderm embryos: (1) formaldehyde fixation of cleavage stage sea urchin embryos, (2) staining and imaging fixed embryos, (3) simultaneous fixation and visualization of the actin and microtubule cytoskeletons, (4) observation of live embryos, (5) 4-D imaging of fluorescent markers in live starfish oocytes, (6) protocol for imaging the dynamics of nuclear lamina during nuclear envelope breakdown, and (7) 4-D imaging of dextran entry during nuclear envelope breakdown (NEBD). Several procedures for fixing early sea urchin embryos with formaldehyde, cold methanol, detergents, and buffers are described. For staining and imaging echinoderm embryo, immunofluorescence is a widely used technique, as there are many commercially available antibodies that will cross-react beautifully with endogenous echinoderm proteins. General protocols for the immunofluorescence of formaldehyde-fixed cells and for staining nucleic acids with various small-molecule dyes are provided in the chapter. The embryos of echinoderms are amenable to live observation, accompanied by time-lapse video microscopy. The different aspects of the cells are emphasized using various imaging techniques, such as Differential Interference Contrast (DIC). Live embryos are easily observed as wet mounts on a standard slide or in a perfusion chamber, but the coverslip prevents direct access to the cells. A variety of chamber slides are described for live observation of echinoderm embryos.

Cytokinesis : New roles for myosin

Myosin II is the motor for cytokinesis, an event at the end of cell division during which the animal cell uses a contractile ring to pinch itself in half. New and surprising research shows that myosin, either through light chain phosphorylation or through its ATPase activity, also plays an important role in both the assembly and disassembly of the actin contractile ring.