Microtubule disassembly delays the G2–M transition in vertebrates

AbstractWhen cell cultures in growth are treated with drugs that cause microtubules to disassemble, the mitotic index (MI) progressively increases as the cells accumulate in a C-mitosis. For many cell types, however, including rat kangaroo kidney PtK1 cells, the MI does not increase during the first several hours of treatment [1–3] (Figure 1). This ‘lag’ implies either that cells are entering mitosis but rapidly escaping the block, or that they are delayed from entering division. To differentiate between these possibilities, we fixed PtK1 cultures 0, 90 and 270 minutes after treatment with nocodazole, colcemid, lumi-colcemid, taxol or cytochalasin D. After 90 minutes, we found that the numbers of prophase cells in cultures treated with nocodazole or colcemid were reduced by ∼80% relative to cultures treated with lumi-colcemid, cytochalasin D or taxol. Thus, destroying microtubules delays late G2 cells from entering prophase and, as the MI does not increase during this time, existing prophase cells do not enter prometaphase. When mid-prophase cells were treated with nocodazole, the majority (70%) decondensed their chromosomes and returned to G2 before re-entering and completing prophase 3–10 hours later. Thus, a pathway exists in vertebrates that delays the G2–M transition when microtubules are disassembled during the terminal stages of G2. As this pathway induces mid-prophase cells to transiently decondense their chromosomes, it is likely that it downregulates the cyclin A–cyclin-dependent kinase 2 (CDK2) complex, which is required in vertebrates for the early stages of prophase [4]

The nature of cell-cycle checkpoints: facts and fallacies

The concept of checkpoint controls revolutionized our understanding of the cell cycle. Here we revisit the defining features of checkpoints and argue that failure to properly appreciate the concept is leading to misinterpretation of experimental results. We illustrate, using the mitotic checkpoint, problems that can arise from a failure to respect strict definitions and precise terminology

Snell's law for surface electrons: Refraction of an electron gas imaged in real space

On NaCl(100)/Cu(111) an interface state band is observed that descends from the surface-state band of the clean copper surface. This band exhibits a Moire-pattern-induced one-dimensional band gap, which is accompanied by strong standing-wave patterns, as revealed in low-temperature scanning tunneling microscopy images. At NaCl island step edges, one can directly see the refraction of these standing waves, which obey Snell's refraction law.Comment: 4 pages, 4 figure

Human resource requirements for quality-assured electronic data capture of the tuberculosis case register

<p>Abstract</p> <p>Background</p> <p>The tuberculosis case register is the data source for the reports submitted by basic management units to the national tuberculosis program. Our objective was to measure the data entry time required to complete and double-enter one record, and to estimate the time for the correction of errors in the captured information from tuberculosis case registers in Cambodia and Viet Nam. This should assist in quantifying the additional requirements in human resources for national programs moving towards electronic recording and reporting.</p> <p>Methods</p> <p>Data from a representative sample of tuberculosis case registers from Cambodia and Viet Nam were double-entered and discordances resolved by rechecking the original case register. Computer-generated data entry time recorded the time elapsed between opening of a new record and saving it to disk.</p> <p>Results</p> <p>The dataset comprised 22,732 double-entered records of 11,366 patients (37.1% from Cambodia and 62.9% from Viet Nam). The mean data entry times per record were 97.5 (95% CI: 96.2-98.8) and 66.2 (95% CI: 59.5-73.0) seconds with medians of 90 and 31 s respectively in Cambodia and in Viet Nam. The percentage of records with an error was 6.0% and 39.0% respectively in Cambodia and Viet Nam. Data entry time was inversely associated with error frequency. We estimate that approximately 118-person-hours were required to produce 1,000 validated records.</p> <p>Conclusions</p> <p>This study quantifies differences between two countries for data entry time for the tuberculosis case register and frequencies of data entry errors and suggests that higher data entry speed is partially offset by requiring revisiting more records for corrections.</p

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle

Imaging the division process in living tissue culture cells

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Methods 38 (2006): 2-16, doi:10.1016/j.ymeth.2005.07.007.We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy

Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied

© 2008 Brito et al. This article is distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 182 (2009): 623-629, doi:10.1083/jcb.200805072.When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.This research was supported by The National Institutes of Health (GMS 40198 to C.L. Rieder) and a doctoral research fellowship (SFRH/ BD/13663/2003) from Fundacao para a Ciencia e a Tecnologia (to D.A. Brito)