Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells.

Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines.

Live-cell imaging reveals square shape spindles and long mitosis duration in the silkworm holocentric cells.

Proper chromosome segregation during mitosis requires both the assembly of a microtubule (MT)-based spindle and the assembly of DNA-centromere-based kinetochore structure. Kinetochore-to-MT attachment enables chromosome separation. Monocentric cells, such as found in human, have one unique kinetochore per chromosome. Holocentric cells, such as found in the silkworm, in contrast, have multiple kinetochore structures per chromosome. Interestingly, some human cancer chromosomes contain more than one kinetochore, a condition called di- and tricentric. Thus, comparing how wild-type mono- and holocentric cells perform mitosis may provide novel insights into cancer di- and tricentric cell mitosis. We present here live-cell imaging of human RPE1 and silkworm BmN4 cells, revealing striking differences in spindle architecture and dynamics, and highlighting differential kinesin function between mono- and holocentric cells.

Fission yeast mtr1p regulates interphase microtubule cortical dwell-time.

The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics.

Studying mitochondria and microtubule localization and dynamics in standardized cell shapes.

Mammalian cells show a large diversity in shape and are both shape-changing and mobile when cultured on conventional uniform substrates. The use of micropatterning techniques limits the number of variable parameters, by imposing shape and standardized adhesive areas on the cells, which facilitates analysis. By changing size or shape of the micropattern, for example, forcing a polar axis on the cell, it is possible to study how these parameters impact organelle organization, distribution, and dynamics inside the cell. To study the mitochondrial network, which is composed of dynamic tubular organelles dependent on the microtubule cytoskeleton for its distribution, it is important to be able to distinguish between distinct mitochondria. Here, we present a practical method with which we spread the cells on large patterns created with deep UV technique, which not only makes the cells uniform in size and shape as well as immobile, and therefore easier to compare and analyze, but also expands the mitochondrial network and allows for an easier tracking of appropriately labeled individual mitochondria.

A fast microfluidic temperature control device for studying microtubule dynamics in fission yeast.

Recent development in soft lithography and microfluidics enables biologists to create tools to control the cellular microenvironment. One such control is the ability to quickly change the temperature of the cells. Genetic model organism such as fission yeast has been useful for studies of the cell cytoskeleton. In particular, the dynamic microtubule cytoskeleton responds to changes in temperature. In addition, there are temperature-sensitive mutations of cytoskeletal proteins. We describe here the fabrication and use of a microfluidic device to quickly and reversibly change cellular temperature between 2 degrees C and 50 degrees C. We demonstrate the use of this device while imaging at high-resolution microtubule dynamics in fission yeast.