scholarly journals Time-Lapse Microscopy Reveals Unique Roles for Kinesins during Anaphase in Budding Yeast

1998 ◽  
Vol 143 (3) ◽  
pp. 687-694 ◽  
Author(s):  
Aaron F. Straight ◽  
John W. Sedat ◽  
Andrew W. Murray
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Dynamic Structure ◽  
Time Lapse ◽  
Slow Phase ◽  
Rapid Phase ◽  
Green Fluorescent ◽  
Anaphase B

The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Δ mutants lack the rapid phase of anaphase B, kip1Δ mutants show defects in the slow phase of anaphase B, and kip3Δ mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Δ and kip1Δ mutants affect the duration of anaphase, but cin8Δ does not.

scholarly journals Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

2000 ◽  
Vol 148 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Marisa Segal ◽  
Duncan J. Clarke ◽  
Paul Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynamic Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

scholarly journals The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

1998 ◽  
Vol 9 (7) ◽  
pp. 1741-1756 ◽  
Author(s):  
Jason A. Kahana ◽  
Gabriel Schlenstedt ◽  
Darren M. Evanchuk ◽  
John R. Geiser ◽  
M. Andrew Hoyt ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Stable Complex ◽  
Complex Protein ◽  
Mother And Daughter ◽  
Green Fluorescent ◽  
Dynactin Complex

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene,NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150 glued . Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

scholarly journals The Role of Microtubules in Rapid Hyphal Tip Growth of Aspergillus nidulans

2005 ◽  
Vol 16 (2) ◽  
pp. 918-926 ◽  
Author(s):  
Tetsuya Horio ◽  
Berl R. Oakley
Keyword(s):  
Growth Rate ◽  
Aspergillus Nidulans ◽  
Tip Growth ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Polarized Growth ◽  
Green Fluorescent ◽  
Tip Cells ◽  
Hyphal Tip

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-α-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew ≈5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10× reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.

scholarly journals Spindle Formation inAspergillusIs Coupled to Tubulin Movement into the Nucleus

2003 ◽  
Vol 14 (5) ◽  
pp. 2192-2200 ◽  
Author(s):  
Yulia Ovechkina ◽  
Paul Maddox ◽  
C. Elizabeth Oakley ◽  
Xin Xiang ◽  
Stephen A. Osmani ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Interphase Nuclei ◽  
Spindle Formation ◽  
Spinning Disk ◽  
Green Fluorescent

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/α-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.

scholarly journals Budding Yeast Chromosome Structure and Dynamics during Mitosis

2001 ◽  
Vol 152 (6) ◽  
pp. 1255-1266 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole ◽  
Elastic Recoil ◽  
Rapid Acquisition ◽  
Anaphase Onset ◽  
Spindle Pole Bodies ◽  
Spindle Elongation ◽  
Green Fluorescent ◽  
Anaphase B

Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

scholarly journals Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts

2001 ◽  
Vol 153 (4) ◽  
pp. 881-888 ◽  
Author(s):  
Karen A. Beningo ◽  
Micah Dembo ◽  
Irina Kaverina ◽  
J. Victor Small ◽  
Yu-li Wang
Keyword(s):  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Time Lapse ◽  
Fibroblast Migration ◽  
Motile Cells ◽  
Green Fluorescent ◽  
Traction Stress

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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