Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

© The Authors, 2009 . This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 186 (2009): 727-738, doi:10.1083/jcb.200902083.Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.This work was supported by a National Institutes of Health grant NIH 38499 to R.D. Vale

Mechanoaccumulative elements of the mammalian actin cytoskeleton

To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a Goldilocks zone must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease

Requirement of the Dynein-Adaptor Spindly for Mitotic and Post-Mitotic Functions in Drosophila

Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila

ALADIN is Required for the Production of Fertile Mouse Oocytes

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome

Aircraft-based inversions quantify the importance of wetlands and livestock for Upper Midwest methane emissions

We apply airborne measurements across three seasons (summer, winter and spring 2017-2018) in a multi-inversion framework to quantify methane emissions from the US Corn Belt and Upper Midwest, a key agricultural and wetland source region. Combing our seasonal results with prior fall values we find that wetlands are the largest regional methane source (32 %, 20 [16-23] Gg/d), while livestock (enteric/manure; 25 %, 15 [14-17] Gg/d) are the largest anthropogenic source. Natural gas/petroleum, waste/landfills, and coal mines collectively make up the remainder. Optimized fluxes improve model agreement with independent datasets within and beyond the study timeframe. Inversions reveal coherent and seasonally dependent spatial errors in the WetCHARTs ensemble mean wetland emissions, with an underestimate for the Prairie Pothole region but an overestimate for Great Lakes coastal wetlands. Wetland extent and emission temperature dependence have the largest influence on prediction accuracy; better representation of coupled soil temperature-hydrology effects is therefore needed. Our optimized regional livestock emissions agree well with the Gridded EPA estimates during spring (to within 7 %) but are g≈ 25 % higher during summer and winter. Spatial analysis further shows good top-down and bottom-up agreement for beef facilities (with mainly enteric emissions) but larger (g≈ 30 %) seasonal discrepancies for dairies and hog farms (with \u3e 40 % manure emissions). Findings thus support bottom-up enteric emission estimates but suggest errors for manure; we propose that the latter reflects inadequate treatment of management factors including field application. Overall, our results confirm the importance of intensive animal agriculture for regional methane emissions, implying substantial mitigation opportunities through improved management

Upscaling Wetland Methane Emissions From the FLUXNET-CH4 Eddy Covariance Network (UpCH4 v1.0):Model Development, Network Assessment, and Budget Comparison

Wetlands are responsible for 20%–31% of global methane (CH4) emissions and account for a large source of uncertainty in the global CH4 budget. Data-driven upscaling of CH4 fluxes from eddy covariance measurements can provide new and independent bottom-up estimates of wetland CH4 emissions. Here, we develop a six-predictor random forest upscaling model (UpCH4), trained on 119 site-years of eddy covariance CH4 flux data from 43 freshwater wetland sites in the FLUXNET-CH4 Community Product. Network patterns in site-level annual means and mean seasonal cycles of CH4 fluxes were reproduced accurately in tundra, boreal, and temperate regions (Nash-Sutcliffe Efficiency ∼0.52–0.63 and 0.53). UpCH4 estimated annual global wetland CH4 emissions of 146 ± 43 TgCH4 y−1 for 2001–2018 which agrees closely with current bottom-up land surface models (102–181 TgCH4 y−1) and overlaps with top-down atmospheric inversion models (155–200 TgCH4 y−1). However, UpCH4 diverged from both types of models in the spatial pattern and seasonal dynamics of tropical wetland emissions. We conclude that upscaling of eddy covariance CH4 fluxes has the potential to produce realistic extra-tropical wetland CH4 emissions estimates which will improve with more flux data. To reduce uncertainty in upscaled estimates, researchers could prioritize new wetland flux sites along humid-to-arid tropical climate gradients, from major rainforest basins (Congo, Amazon, and SE Asia), into monsoon (Bangladesh and India) and savannah regions (African Sahel) and be paired with improved knowledge of wetland extent seasonal dynamics in these regions. The monthly wetland methane products gridded at 0.25° from UpCH4 are available via ORNL DAAC (https://doi.org/10.3334/ORNLDAAC/2253).</p

ECOSTRESS: NASA's next generation mission to measure evapotranspiration from the International Space Station

The ECOsystem Spaceborne Thermal Radiometer Experiment on Space Station ECOSTRESS) was launched to the International Space Station on June 29, 2018. The primary science focus of ECOSTRESS is centered on evapotranspiration (ET), which is produced as level‐3 (L3) latent heat flux (LE) data products. These data are generated from the level‐2 land surface temperature and emissivity product (L2_LSTE), in conjunction with ancillary surface and atmospheric data. Here, we provide the first validation (Stage 1, preliminary) of the global ECOSTRESS clear‐sky ET product (L3_ET_PT‐JPL, version 6.0) against LE measurements at 82 eddy covariance sites around the world. Overall, the ECOSTRESS ET product performs well against the site measurements (clear‐sky instantaneous/time of overpass: r2 = 0.88; overall bias = 8%; normalized RMSE = 6%). ET uncertainty was generally consistent across climate zones, biome types, and times of day (ECOSTRESS samples the diurnal cycle), though temperate sites are over‐represented. The 70 m high spatial resolution of ECOSTRESS improved correlations by 85%, and RMSE by 62%, relative to 1 km pixels. This paper serves as a reference for the ECOSTRESS L3 ET accuracy and Stage 1 validation status for subsequent science that follows using these data

Upscaling Wetland Methane Emissions From the FLUXNET-CH4 Eddy Covariance Network (UpCH4 v1.0): Model Development, Network Assessment, and Budget Comparison

Wetlands are responsible for 20%-31% of global methane (CH4) emissions and account for a large source of uncertainty in the global CH4 budget. Data-driven upscaling of CH4 fluxes from eddy covariance measurements can provide new and independent bottom-up estimates of wetland CH4 emissions. Here, we develop a six-predictor random forest upscaling model (UpCH4), trained on 119 site-years of eddy covariance CH4 flux data from 43 freshwater wetland sites in the FLUXNET-CH4 Community Product. Network patterns in site-level annual means and mean seasonal cycles of CH4 fluxes were reproduced accurately in tundra, boreal, and temperate regions (Nash-Sutcliffe Efficiency similar to 0.52-0.63 and 0.53). UpCH(4) estimated annual global wetland CH4 emissions of 146 +/- 43 TgCH4 y(-1) for 2001-2018 which agrees closely with current bottom-up land surface models (102-181 TgCH4 y(-1)) and overlaps with top-down atmospheric inversion models (155-200 TgCH4 y -1). However, UpCH4 diverged from both types of models in the spatial pattern and seasonal dynamics of tropical wetland emissions. We conclude that upscaling of eddy covariance CH4 fluxes has the potential to produce realistic extra-tropical wetland CH4 emissions estimates which will improve with more flux data. To reduce uncertainty in upscaled estimates, researchers could prioritize new wetland flux sites along humid-to-arid tropical climate gradients, from major rainforest basins (Congo, Amazon, and SE Asia), into monsoon (Bangladesh and India) and savannah regions (African Sahel) and be paired with improved knowledge of wetland extent seasonal dynamics in these regions. The monthly wetland methane products gridded at 0.25 degrees from UpCH4 are available via ORNL DAAC (https://doi.org/10.3334/ ORNLDAAC/2253).Plain Language Summary Wetlands account for a large share of global methane emissions to the atmosphere, but current estimates vary widely in magnitude (similar to 30% uncertainty on annual global emissions) and spatial distribution, with diverging predictions for tropical rice growing (e.g., Bengal basin), rainforest (e.g., Amazon basin), and floodplain savannah (e.g., Sudd) regions. Wetland methane model estimates could be improved by increased use of land surface methane flux data. Upscaling approaches use flux data collected across globally distributed measurement networks in a machine learning framework to extrapolate fluxes in space and time. Here, we train and evaluate a methane upscaling model (UpCH4) and use it to generate monthly, globally gridded wetland methane emissions estimates for 2001-2018. The UpCH4 model uses only six predictor variables among which temperature is dominant. Global annual methane emissions estimates and associated uncertainty ranges from upscaling fall within state-of-the-art model ensemble estimates from the Global Carbon Project (GCP) methane budget. In some tropical regions, the spatial pattern of UpCH4 emissions diverged from GCP predictions, however, inclusion of flux measurements from additional ground-based sites, together with refined maps of tropical wetlands extent, could reduce these prediction uncertainties

Correction: Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype–phenotype correlation

n/