Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pelletier, J. F., Field, C. M., Furthauer, S., Sonnett, M., & Mitchison, T. J. Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm. Elife, 9, (2020): e60047, https://doi.org/10.7554/eLife.60047.How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed that dynein-based, inward organelle transport generates length-dependent pulling forces that move centrosomes and MTs outwards, while other components of cytoplasm are static. We imaged aster movement by dynein and actomyosin forces in Xenopus egg extracts and observed outward co-movement of MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and soluble fluorescein. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster periphery, then mostly halted inside the aster, while dynein-coated beads moved to the aster center at a constant rate, suggesting organelle movement is limited by brake proteins or other sources of drag. These observations call for new models in which all components of the cytoplasm comprise a mechanically integrated aster gel that moves collectively in response to dynein and actomyosin forces.This work was supported by NIH grant R35GM131753 (TJM) and MBL fellowships from the Evans Foundation, MBL Associates, and the Colwin Fund (TJM and CMF). JFP was supported by the Fannie and John Hertz Foundation, the Fakhri lab at MIT, the MIT Department of Physics, and the MIT Center for Bits and Atoms

Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin

The chromosomal passenger complex (CPC) is a conserved, essential regulator of cell division. As such, significant anti–cancer drug development efforts have been focused on targeting it, most notably by inhibiting its AURKB kinase subunit. The CPC is activated by AURKB-catalyzed autophosphorylation on multiple subunits, but how this regulates CPC interactions with other mitotic proteins remains unclear. We investigated the hydrodynamic behavior of the CPC in Xenopus laevis egg cytosol using sucrose gradient sedimentation and in HeLa cells using fluorescence correlation spectroscopy. We found that autophosphorylation of the CPC decreases its sedimentation coefficient in egg cytosol and increases its diffusion coefficient in live cells, indicating a decrease in mass. Using immunoprecipitation coupled with mass spectrometry and immunoblots, we discovered that inactive, unphosphorylated CPC interacts with nucleophosmin/nucleoplasmin proteins, which are known to oligomerize into pentamers and decamers. Autophosphorylation of the CPC causes it to dissociate from nucleophosmin/nucleoplasmin. We propose that nucleophosmin/nucleoplasmin complexes serve as chaperones that negatively regulate the CPC and/or stabilize its inactive form, preventing CPC autophosphorylation and recruitment to chromatin and microtubules in mitosis

The Main Belt Comets and ice in the Solar System

We review the evidence for buried ice in the asteroid belt; specifically the questions around the so-called Main Belt Comets (MBCs). We summarise the evidence for water throughout the Solar System, and describe the various methods for detecting it, including remote sensing from ultraviolet to radio wavelengths. We review progress in the first decade of study of MBCs, including observations, modelling of ice survival, and discussion on their origins. We then look at which methods will likely be most effective for further progress, including the key challenge of direct detection of (escaping) water in these bodies

Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

Quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g., TMT) has enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice, existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approach (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time, the median coefficient of variation improves from 13% to 4%. Thus, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments

Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

Quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g., TMT) has enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice, existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approach (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time, the median coefficient of variation improves from 13% to 4%. Thus, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments

Ground State Destabilization from a Positioned General Base in the Ketosteroid Isomerase Active Site

We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction’s Michaelis complex. The analogue binding affinity increased ∼1 order of magnitude for the Asp38Asn mutation and ∼2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Asp general base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids