Colloid osmotic parameterization and measurement of subcellular crowding

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mitchison, T. J. (2019). Colloid osmotic parameterization and measurement of subcellular crowding. Molecular Biology of the Cell, 30(2), (2019): 173-180, doi:10.1091/mbc.E18-09-0549.Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1–2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration–pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure–tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.This article was prompted by lively discussions at the Marine Biological Laboratory (MBL) Physiology Course, Woods Hole, MA. I particularly thank Annie Pipathsouk (University of California, San Franscico) and Charlotte Strandkvist (Harvard Medical School) for experimental work in frog egg extract; James Pelletier (MIT), Tony Hyman (MPI Dresden), and Rob Phillips (Cal. Tech.) for discussions; and Nikon for microscopy support at MBL. T.J.M. is supported by National Institute of General Medical Sciences 39565

Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol

BACKGROUND: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain. RESULTS: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure. CONCLUSION: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5

Force and length in the mitotic spindle

Author Posting. © The Author(s), 2009. 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 Current Biology 19 (2009): R749-R761, doi:10.1016/j.cub.2009.07.028.The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly-disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force producing mechanisms. We then discuss models for force integration, and spindle length determination. We also emphasize key missing data that notably includes absolute values of forces, and how they vary as a function of position, within the spindle.S.D. received support from a Milton Fund (Harvard University) and T.J.M. was supported by NIH grants GM039565 and P50 GM068763

Meiotic Spindle: Sculpted by Severing

Katanin is a conserved AAA ATPase with the ability to sever microtubules, but its biological function in animal cells has been obscure. A recent study using electron tomography has found that katanin stimulates the production of microtubules in the meiotic spindles of Caenorhabditis elegans oocytes

Vascular Disrupting Agent Drug Classes Differ in Effects on the Cytoskeleton

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function

Mitosis: New Roles for Myosin-X and Actin at the Spindle

SummaryRoles for actin and myosin in positioning mitotic spindles in the cell are well established. A recent study of myosin-X function in early Xenopus embryo mitosis now reports that this unconventional myosin is required for pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle

Maintenance of Mitochondrial Oxygen Homeostasis by Cosubstrate Compensation

Mitochondria maintain a constant rate of aerobic respiration over a wide range of oxygen levels. However, the control strategies underlying oxygen homeostasis are still unclear. Using mathematical modeling, we found that the mitochondrial electron transport chain (ETC) responds to oxygen level changes by undergoing compensatory changes in reduced electron carrier levels. This emergent behavior, which we named cosubstrate compensation (CSC), enables the ETC to maintain homeostasis over a wide of oxygen levels. When performing CSC, our ETC models recapitulated a classic scaling relationship discovered by Chance [Chance B (1965) J. Gen. Physiol. 49:163-165] relating the extent of oxygen homeostasis to the kinetics of mitochondrial electron transport. Analysis of an in silico mitochondrial respiratory system further showed evidence that CSC constitutes the dominant control strategy for mitochondrial oxygen homeostasis during active respiration. Our findings indicate that CSC constitutes a robust control strategy for homeostasis and adaptation in cellular biochemical networks

Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 29 (2018): 304-316, doi:10.1091/mbc.E17-09-0540.The cleavage furrow in Xenopus zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the midplane, they assemble a disk-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin that instructs the cleavage furrow. Here we investigate the mechanism that keeps the two asters separate and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg orthologue of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in Xenopus egg extracts. Prc1E was required for Kif4A recruitment but not vice versa. Microtubule plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote “anti-parallel pruning” that enforces radial organization within asters and generates boundaries to microtubule growth between asters