The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is ...determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.
The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, ...flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin–F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin–F-actin networks.
MYH9‐related disease (MYH9‐RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin‐activated motor protein non‐muscle myosin IIA (NMIIA). MYH9‐RD patients ...suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA‐expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F‐actin) in the spectrin‐F‐actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled‐coil rod, or non‐helical tail) on RBC NMIIA function. We found that MYH9‐RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron‐scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9‐RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F‐actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub‐clinical phenotype of MYH9‐RD.
Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain ...antiretrovirals (ARVs) may contribute to HAND.
We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis.
Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after 7 days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults.
This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.
In convergent evolution, similar phenotypes evolve repeatedly in independent populations, often reflecting adaptation to similar environments. Understanding whether convergent evolution proceeds via ...similar or different genetic and developmental mechanisms offers insight towards the repeatability and predictability of evolution. Oceanic populations of threespine stickleback fish, Gasterosteus aculeatus, have repeatedly colonized countless freshwater lakes and streams, where new diets lead to morphological adaptations related to feeding. Here, we show that heritable increases in branchial bone length have convergently evolved in two independently derived freshwater stickleback populations. In both populations, an increased bone growth rate in juveniles underlies the convergent adult phenotype, and one population also has a longer cartilage template. Using F2 crosses from these two freshwater populations, we show that two quantitative trait loci (QTL) control branchial bone length at distinct points in development. In both populations, a QTL on chromosome 21 controls bone length throughout juvenile development, and a QTL on chromosome 4 controls bone length only in adults. In addition to these similar developmental profiles, these QTL show similar chromosomal locations in both populations. Our results suggest that sticklebacks have convergently evolved longer branchial bones using similar genetic and developmental programmes in two independently derived populations.
In convergent evolution, similar phenotypes evolve repeatedly in independent populations, often reflecting adaptation to similar environments. Understanding whether convergent evolution proceeds via ...similar or different genetic and developmental mechanisms offers insight towards the repeatability and predictability of evolution. Oceanic populations of three-spine stickleback fish, Gasterosteus aculeatus, have repeatedly colonized countless freshwater lakes and streams, where new diets lead to morphological adaptations related to feeding. Here, we show that heritable increases in branchial bone length have convergently evolved in two independently derived freshwater stickleback populations. In both populations, an increased bone growth rate in juveniles underlies the convergent adult phenotype, and one population also has a longer cartilage template. Using F₂ crosses from these two freshwater populations, we show that two quantitative trait loci (QTL) control branchial bone length at distinct points in development. In both populations, a QTL on chromosome 21 controls bone length throughout juvenile development, and a QTL on chromosome 4 controls bone length only in adults. In addition to these similar developmental profiles, these QTL show similar chromosomal locations in both populations. Our results suggest that sticklebacks have convergently evolved longer branchial bones using similar genetic and developmental programmes in two independently derived populations.
The biconcave disk shape, deformability, and resiliency of the mammalian red blood cell (RBC) are vital to its circulatory function. These features rely on the membrane skeleton, a viscoelastic ...network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. RBCs contain nonmuscle myosin II (NMII), an F-actin-activated ATPase that exerts force on F-actin networks in many cells types to control cell shapes and membrane properties. However, a function for NMII contractility in RBCs has not previously been tested. The work presented in this thesis shows that nonmuscle myosin IIA (NMIIA) forms bipolar filaments that interact with the membrane skeleton to control RBC membrane curvature and deformability. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to F-actin, and the phosphorylation of RBC NMIIA heavy and light chains indicate active regulation of NMIIA motor activity and filament assembly. Inhibition of NMII motor activity with blebbistatin demonstrates a role for this activity in NMIIA-membrane association, nanoscale membrane oscillations, membrane curvature, and RBC deformability. Characterization of RBC phenotypes in patients with NMIIA heavy chain mutations shows that patients with motor domain mutations have the most severe, phenotypes, further supporting the role of this domain in RBC NMIIA function. While patients do not have clinically significant anemias, patient RBCs exhibit abnormal morphologies and hematological indices. Some mutations affect NMIIA association with the membrane skeleton and regulation of filament assembly through heavy chain phosphorylation, likely contributing to these phenotypes. Together, these data indicate a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these results demonstrate a general function for NMII in controlling membrane morphology and mechanical properties through contractile interactions with spectrin-F-actin networks.
Objective Little is known regarding the impact of mode of delivery in the periviable period. Even less is understood regarding the effect of mode of delivery on neurodevelopment. Our objective is to ...determine if the mode of delivery at time of periviability impacts Bayley II scores at 2 years of age. Study Design This is a secondary analysis of a randomized, controlled trial of magnesium sulfate for the prevention of cerebral palsy, a multicenter trial where women at imminent risk for delivery were assigned to receive magnesium sulfate or placebo. For this secondary analysis we included nonanomalous singleton gestations delivered between 23 4/7 and 25 6/7 weeks. We excluded women with missing exposure or outcome data. The primary exposure of interest was mode of delivery. The primary outcome was Bayley II scores <70 (mental and motor) at 2 years of age. Log binomial regression was used to control for possible confounders including gestational age at delivery, presentation at time of delivery, chorioamnionitis, years of maternal education, maternal body mass index, and original study treatment group. Results A total of 158 women met inclusion criteria. In all, 91 had a vaginal delivery and 67 had a cesarean delivery. Exposure to magnesium sulfate, maternal education, chorioamnionitis, years of maternal education, and maternal body mass index were similar in both groups. There was no difference in either mental or motor Bayley II scores <70 or <85 by mode of delivery in either univariable or multivariable analysis. Conclusion There is no detectable difference in Bayley II scores between mode of delivery at time of periviability. This adds to the literature supporting obstetric indications dictating mode of delivery at this gestational age.
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