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

Systems Biochemistry of the Metaphase Spindle

Our research aims were motivated by a desire to understand the complexity of living self-organized systems. We focused on microtubule-based systems in their entirety, and thus were led to mass spectrometry-based proteomics as our measurement approach of choice. We made multiple improvements to various aspects of this measurement pipeline. We developed a new quantitative proteomics measurement approach that has significantly better signal to noise (median of >100) than the previous state-of-the-art (~30). We also developed a novel computational approach for imputing missing values in large quantitative proteomics datasets. Our approach relies on coupling underlying biological covariation between samples with regularized regression. We also investigated the properties of matching acquired mass spectra to peptide sequences with database searches that varied widely in the size of the precursor mass error used. We found that using search spaces that are ~250 times larger than what is typically used confer multiple advantages and can increase the potential number of sequence matches by 20 to 35% in various datasets. We used advances in proteomics methodology from ourselves and others to do some biology. We used quantitative multiplexed proteomics to obtain a systems biochemistry view of microtubule based structures in cell free extracts from the model system X. laevis. By combining classical biochemical binding assays with modern mass spectrometry we were able to measure partition coefficients for microtubules and chromatin for thousands of proteins. In some cases, exchange rates and salt sensitivities or other proxies for affinity were also measured. We developed and used a rapid filtration approach to isolate metaphase spindles directly from X. laevis extracts faithfully and measure their protein composition.Chemical Biolog

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

Co-movement of astral microtubules, organelles and F-actin suggests aster positioning by surface forces in frog eggs

AbstractHow bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed dynein-based organelle transport generates length-dependent forces on astral MTs that pull centrosomes through the cytoplasm, away from the midplane. In Xenopus egg extracts, we co-imaged MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and fluorescein in moving and stationary asters. In asters that were moving in response to dynein and actomyosin forces, we observed that all cytoplasmic components moved together, i.e., as a continuum. Dynein-mediated organelle transport was restricted by interior MTs and F-actin. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster surface, then mostly halted inside the aster. Dynein-coated beads were slowed by F-actin, but in contrast to organelles, beads did not halt inside asters. These observations call for new models of aster positioning based on surface forces and internal stresses.</jats:p

One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses

AbstractThe management of pandemics such as COVID-19 requires highly scalable and sensitive viral diagnostics, together with variant identification. Next-generation sequencing (NGS) has many attractive features for highly multiplexed testing, however current sequencing-based methods are limited in throughput by early processing steps on individual samples (e.g. RNA extraction and PCR amplification). Here we report a new method, “One-Seq”, that eliminates the current bottlenecks in scalability by enabling early pooling of samples, before any extraction or amplification steps. To enable early pooling, we developed a one-pot reaction for efficient reverse transcription (RT) and upfront barcoding in extraction-free clinical samples, and a “protector” strategy in which carefully designed competing oligonucleotides prevent barcode crosstalk and preserve detection of the high dynamic range of viral load in clinical samples. This method is highly sensitive, achieving a limit of detection (LoD) down to 2.5 genome copy equivalent (gce) in contrived RT samples, 10 gce in multiplexed sequencing, and 2-5 gce with multi-primer detection, suggesting an LoD of 200-500 gce/ml for clinical testing. In clinical specimens, One-Seq showed quantitative viral detection against clinical Ct values with 6 logs of linear dynamic range and detection of SARS-CoV-2 positive samples down to ∼360 gce/ml. In addition, One-Seq reports a number of hotspot viral mutations at equal scalability at no extra cost. Scaling up One-Seq would allow a throughput of 100,000-1,000,000 tests per day per single clinical lab, at an estimated amortized reagent cost of $1.5 per test and turn-around time of 7.5-15 hr.</jats:p