Elevated Blood Pressure in Pregnancy and Long-Term Cardiometabolic Health Outcomes.

OBJECTIVE: To examine the association between elevated blood pressure (BP) in the early third trimester and cardiometabolic health 10-14 years after delivery. METHODS: This is a secondary analysis from the prospective HAPO FUS (Hyperglycemia and Adverse Pregnancy Outcome Follow-Up Study). Blood pressure in the early third trimester was categorized per American College of Cardiology/American Heart Association thresholds for: normal BP below 120/80 mm Hg (reference), elevated BP 120-129/below 80 mm Hg, stage 1 hypertension 130-139/80-89 mm Hg, and stage 2 hypertension 140/90 mm Hg or higher. Cardiometabolic outcomes assessed 10-14 years after the index pregnancy were type 2 diabetes mellitus and measures of dyslipidemia, including low-density lipoprotein (LDL) cholesterol 130 mg/dL or higher, total cholesterol 200 mg/dL or higher, high-density lipoprotein (HDL) cholesterol 40 mg/dL or lower, and triglycerides 200 mg/dL or higher. Adjusted analysis was performed with the following covariates: study field center, follow-up duration, age, body mass index (BMI), height, family history of hypertension and diabetes, smoking and alcohol use, parity, and oral glucose tolerance test glucose z score. RESULTS: Among 4,692 pregnant individuals at a median gestational age of 27.9 weeks (interquartile range 26.6-28.9 weeks), 8.5% (n=399) had elevated BP, 14.9% (n=701) had stage 1 hypertension, and 6.4% (n=302) had stage 2 hypertension. At a median follow-up of 11.6 years, among individuals with elevated BP, there was a higher frequency of diabetes (elevated BP: adjusted relative risk [aRR] 1.88, 95% CI, 1.06-3.35; stage 1 hypertension: aRR 2.58, 95% CI, 1.62-4.10; stage 2 hypertension: aRR 2.83, 95% CI, 1.65-4.95) compared with those with normal BP. Among individuals with elevated BP, there was a higher frequency of elevated LDL cholesterol (elevated BP: aRR 1.27, 95% CI, 1.03-1.57; stage 1 hypertension: aRR 1.22, 95% CI, 1.02-1.45, and stage 2 hypertension: aRR 1.38, 95% CI, 1.10-1.74), elevated total cholesterol (elevated BP: aRR 1.27, 95% CI, 1.07-1.52; stage 1 hypertension: aRR 1.16, 95% CI, 1.00-1.35; stage 2 hypertension: aRR 1.41 95% CI, 1.16-1.71), and elevated triglycerides (elevated BP: aRR 2.24, 95% CI, 1.42-3.53; stage 1 hypertension: aRR 2.15, 95% CI, 1.46-3.17; stage 2 hypertension: aRR 3.24, 95% CI, 2.05-5.11) but not of low HDL cholesterol. CONCLUSION: The frequency of adverse cardiometabolic outcomes at 10-14 years after delivery was progressively higher among pregnant individuals with BP greater than 120/80 in the early third trimester.

Community-level social determinants of health and pregestational and gestational diabetes.

BACKGROUND: Individual adverse social determinants of health are associated with increased risk of diabetes in pregnancy, but the relative influence of neighborhood or community-level social determinants of health is unknown. OBJECTIVE: This study aimed to determine whether living in neighborhoods with greater socioeconomic disadvantage, food deserts, or less walkability was associated with having pregestational diabetes and developing gestational diabetes. STUDY DESIGN: We conducted a secondary analysis of the prospective Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-To-Be. Home addresses in the first trimester were geocoded at the census tract level. The exposures (modeled separately) were the following 3 neighborhood-level measures of adverse social determinants of health: (1) socioeconomic disadvantage, defined by the Area Deprivation Index and measured in tertiles from the lowest tertile (ie, least disadvantage [T1]) to the highest (ie, most disadvantage [T3]); (2) food desert, defined by the United States Department of Agriculture Food Access Research Atlas (yes/no by low income and low access criteria); and (3) less walkability, defined by the Environmental Protection Agency National Walkability Index (most walkable score [15.26-20.0] vs less walkable score [<15.26]). Multinomial logistic regression was used to model the odds of gestational diabetes or pregestational diabetes relative to no diabetes as the reference, adjusted for age at delivery, chronic hypertension, Medicaid insurance status, and low household income (<130% of the US poverty level). RESULTS: Among the 9155 assessed individuals, the mean Area Deprivation Index score was 39.0 (interquartile range, 19.0-71.0), 37.0% lived in a food desert, and 41.0% lived in a less walkable neighborhood. The frequency of pregestational and gestational diabetes diagnosis was 1.5% and 4.2%, respectively. Individuals living in a community in the highest tertile of socioeconomic disadvantage had increased odds of entering pregnancy with pregestational diabetes compared with those in the lowest tertile (T3 vs T1: 2.6% vs 0.8%; adjusted odds ratio, 2.52; 95% confidence interval, 1.41-4.48). Individuals living in a food desert (4.8% vs 4.0%; adjusted odds ratio, 1.37; 95% confidence interval, 1.06-1.77) and in a less walkable neighborhood (4.4% vs 3.8%; adjusted odds ratio, 1.33; 95% confidence interval, 1.04-1.71) had increased odds of gestational diabetes. There was no significant association between living in a food desert or a less walkable neighborhood and pregestational diabetes, or between socioeconomic disadvantage and gestational diabetes. CONCLUSION: Nulliparous individuals living in a neighborhood with higher socioeconomic disadvantage were at increased odds of entering pregnancy with pregestational diabetes, and those living in a food desert or a less walkable neighborhood were at increased odds of developing gestational diabetes, after controlling for known covariates.

Association between glycemic control and group B streptococcus colonization among pregnant individuals with pregestational diabetes.

PROBLEM: Pregestational diabetes increases the risk of group B streptococcus (GBS) colonization in pregnancy. Whether glycemic control is associated with differences in this risk is unknown. We examined the association between glycemic control and GBS colonization among pregnant individuals with pregestational diabetes. METHOD OF STUDY: A retrospective cohort of pregnant individuals with pregestational diabetes at a tertiary care center. The exposure was glycemic control, measured as hemoglobin A1c (A1c) at >20 weeks and assessed categorically at thresholds of <6.5% and <6.0%, and secondarily, as a continuous percentage. The outcome was maternal GBS colonization. Multivariable logistic regression was used and adjusted for age, parity, race, and ethnicity as a social determinant, body mass index, type of diabetes, and gestational age at A1c assessment. RESULTS: Among 305 individuals (33% Type 1, 67% type 2), 45.0% (n = 140) were colonized with GBS. Individuals with an A1c < 6.5% were half as likely to be colonized with GBS compared with those with a A1c ≥ 6.5% (38.8% vs. 53.9%; adjusted odds ratio, AOR: 0.55; 95% CI: 0.33-0.91). Results were unchanged at an A1c threshold of <6.0% (35.7% vs. 48.5%; AOR: 0.60; 95% CI: 0.36-0.98). Individuals with a higher A1c as a continuous measure (%) were more likely to be colonized (AOR: 1.57 per 1%; 95% CI: 1.25-1.97). CONCLUSIONS: Pregnant individuals with pregestational diabetes with worse glycemic control were at an increased risk of GBS colonization. Further study is needed to understand if improved glycemic control leads to lower risk of GBS colonization.

Challenges in Conducting Therapeutic Trials in Pregnancy: Emphasizing Recent Lessons Learned.

Pregnant people have traditionally been excluded from therapeutic research by restrictions intended for fetal protection. Despite a movement toward inclusion, concerns for the feasibility and safety of including pregnant people in studies continue to limit this research. This article reviews the history of research guidelines in pregnancy and illustrates ongoing challenges, as seen in the development of vaccines and therapies during the coronavirus disease 2019 pandemic and investigation of statins for preeclampsia prevention. It explores new approaches that may be used to improve therapeutic research in pregnancy. A major cultural shift is needed to balance potential maternal and/or fetal risks with potential benefits from participation in research, as well as harm from withholding treatment or providing one that is not evidence-based. Finally, it is important to honor maternal autonomy in decision-making regarding participation in clinical trials.

Association of community walkability and glycemic control among pregnant individuals with pregestational diabetes mellitus.

BACKGROUND: Neighborhood walkability is a community-level social determinant of health that measures whether people who live in a neighborhood walk as a mode of transportation. Whether neighborhood walkability is associated with glycemic control among pregnant individuals with pregestational diabetes remains to be defined. OBJECTIVE: This study aimed to evaluate the association between community-level neighborhood walkability and glycemic control as measured by hemoglobin A1c (A1C) among pregnant individuals with pregestational diabetes. STUDY DESIGN: This was a retrospective analysis of pregnant individuals with pregestational diabetes enrolled in an integrated prenatal and diabetes care program from 2012 to 2016. Participant addresses were geocoded and linked at the census-tract level. The exposure was community walkability, defined by the US Environmental Protection Agency National Walkability Index (score range 1-20), which incorporates intersection density (design), proximity to transit stops (distance), and a mix of employment and household types (diversity). Individuals from neighborhoods that were the most walkable (score, 15.26-20.0) were compared with those from neighborhoods that were less walkable (score <15.26), as defined per national Environmental Protection Agency recommendations. The outcomes were glycemic control, including A1C <6.0% and <6.5%, measured both in early and late pregnancy, and mean change in A1C across pregnancy. Modified Poisson regression and linear regression were used, respectively, and adjusted for maternal age, body mass index at delivery, parity, race and ethnicity as a social determinant of health, insurance status, baseline A1C, gestational age at A1C measurement in early and late pregnancy, and diabetes type. RESULTS: Among 417 pregnant individuals (33% type 1, 67% type 2 diabetes mellitus), 10% were living in the most walkable communities. All 417 individuals underwent A1C assessment in early pregnancy (median gestational age, 9.7 weeks; interquartile range, 7.4-14.1), and 376 underwent another A1C assessment in late pregnancy (median gestational age, 30.4 weeks; interquartile range, 27.8-33.6). Pregnant individuals living in the most walkable communities were more likely to have an A1C <6.0% in early pregnancy (15% vs 8%; adjusted relative risk, 1.46; 95% confidence interval, 1.00-2.16), and an A1C <6.5% in late pregnancy compared with those living in less walkable communities (13% vs 9%; adjusted relative risk, 1.33; 95% confidence interval, 1.08-1.63). For individuals living in the most walkable communities, the median A1C was 7.5 (interquartile range, 6.0-9.4) in early pregnancy and 5.9 (interquartile range, 5.4-6.4) in late pregnancy. For those living in less walkable communities, the median A1C was 7.3 (interquartile range, 6.2-9.2) in early pregnancy and 6.2 (interquartile range, 5.6-7.1) in late pregnancy. Change in A1C across pregnancy was not associated with walkability. CONCLUSION: Pregnant individuals with pregestational diabetes mellitus living in more walkable communities had better glycemic control in both early and late pregnancy. Whether community-level interventions to enhance neighborhood walkability can improve glycemic control in pregnancy requires further study.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands.

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Rho and F-actin self-organize within an artificial cell cortex.

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division.1,2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability."3-7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho.7,8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation.9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics.10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo.7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.

Self-Organization of Cellular Units.

The purpose of this review is to explore self-organizing mechanisms that pattern microtubules (MTs) and spatially organize animal cell cytoplasm, inspired by recent experiments in frog egg extract. We start by reviewing conceptual distinctions between self-organizing and templating mechanisms for subcellular organization. We then discuss self-organizing mechanisms that generate radial MT arrays and cell centers in the absence of centrosomes. These include autocatalytic MT nucleation, transport of minus ends, and nucleation from organelles such as melanosomes and Golgi vesicles that are also dynein cargoes. We then discuss mechanisms that partition the cytoplasm in syncytia, in which multiple nuclei share a common cytoplasm, starting with cytokinesis, when all metazoan cells are transiently syncytial. The cytoplasm of frog eggs is partitioned prior to cytokinesis by two self-organizing modules, protein regulator of cytokinesis 1 (PRC1)-kinesin family member 4A (KIF4A) and chromosome passenger complex (CPC)-KIF20A. Similar modules may partition longer-lasting syncytia, such as early Drosophila embryos. We end by discussing shared mechanisms and principles for the MT-based self-organization of cellular units.

Is a Postvoid Residual Necessary? A Randomized Trial of Two Postoperative Voiding Protocols.

OBJECTIVES: This study aimed to compare a backfill-assisted voiding trial (VT) with and without a postvoid residual (PVR) after pelvic reconstructive surgery. METHODS: This was a nonblinded randomized controlled trial of women undergoing pelvic organ prolapse and/or stress incontinence surgery. Participants were randomized immediately after surgery to either a PVR VT or a PVR-free VT. Our primary outcome was the rate of VT failure at discharge. Secondary outcomes included days of catheterization, urinary tract infection (UTI), and prolonged voiding dysfunction. With a power of 80% and an α of 0.05, we needed 126 participants to detect a 25% difference in VT failure (60% in PVR VT vs 35% in PVR-free VT). RESULTS: Participants were enrolled from March 2017 to October 2017. Of the 150 participants, mean age was 59 years, and 33% underwent vaginal hysterectomy, 48% underwent anterior repair, and 75% underwent midurethral sling. Seventy-five (50%) were randomized to PVR VT and 75 (50%) to PVR-free VT, with no differences in baseline demographic or intraoperative characteristics between the 2 groups. Our primary outcome, VT failure, was not significantly different (53% PVR VT vs 53% PVR-free VT, P = 1.0). There were no significant differences in days of postoperative catheterization (1 [0, 4] in PVR VT vs 1 [0, 4] in PVR-free VT, P = 0.90), UTI (20% PVR VT vs 20% PVR-free VT, P = 1.0), or postoperative voiding dysfunction (4% PVR VT vs 5% PVR-free VT, P = 1.0). CONCLUSIONS: When performing a backfill-assisted VT, checking a PVR does not affect VT failure, postoperative duration of catheterization, UTI, or voiding dysfunction.

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

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.

Implementation and Initial Construct Validity Evidence of a Tool, myTIPreport, for Interactive Workplace Feedback on ACGME Milestones.

OBJECTIVE: To describe implementation of myTIPreport for milestone feedback and to initiate construct validity testing of myTIPreport for milestones. DESIGN: myTIPreport was used to provide workplace feedback on Accreditation Council for Graduate Medical Education required milestone sets. Performance of senior learners (postgraduate year [PGY]-4s) was compared to that of junior learners (PGY-1s) to begin the process of construct validity testing for myTIPreport. SETTING: A convenience-based site selection of Obstetrics and Gynecology (OBGYN) residency programs. PARTICIPANTS: OBGYN residents and faculty. RESULTS: Amongst the 12 participating OBGYN residency programs, there were 444 unique learners and 343 unique faculty teachers. A total of 5293 milestone feedback encounters were recorded. Mean PGY-4 performance was rated higher than mean PGY-1 performance on all 25 of the compared milestone sets, with statistically significant differences seen for 19 (76%) of these 25 milestone sets and nonsignificant differences in the predicted direction observed for the other 6 milestone sets. CONCLUSIONS: myTIPreport detected differences between senior and junior learners for the majority of compared feedback encounters for OBGYN residents. Findings support the emerging construct validity of myTIPreport for milestone feedback.

Unconventional Cell Division Cycles from Marine-Derived Yeasts.

Fungi have been found in every marine habitat that has been explored; however, the diversity and functions of fungi in the ocean are poorly understood. In this study, fungi were cultured from the marine environment in the vicinity of Woods Hole, MA, USA, including from plankton, sponge, and coral. Our sampling resulted in 35 unique species across 20 genera. We observed many isolates by time-lapse, differential interference contrast (DIC) microscopy and analyzed modes of growth and division. Several black yeasts displayed highly unconventional cell division cycles compared to those of traditional model yeast systems. Black yeasts have been found in habitats inhospitable to other life and are known for halotolerance, virulence, and stress resistance. We find that this group of yeasts also shows remarkable plasticity in terms of cell size control, modes of cell division, and cell polarity. Unexpected behaviors include division through a combination of fission and budding, production of multiple simultaneous buds, and cell division by sequential orthogonal septations. These marine-derived yeasts reveal alternative mechanisms for cell division cycles that seem likely to expand the repertoire of rules established from classic model system yeasts.

Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs.

The large length scale of Xenopus laevis eggs facilitates observation of bulk cytoplasm dynamics far from the cortex during cytokinesis. The first furrow ingresses through the egg midplane, which is demarcated by chromosomal passenger complex (CPC) localized on microtubule bundles at the boundary between asters. Using an extract system, we found that local kinase activity of the Aurora B kinase (AURKB) subunit of the CPC caused disassembly of F-actin and keratin between asters and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ∼40 µm without microtubules and much farther with microtubules present. Consistent with extract observations, we observed disassembly of the keratin network between asters in zygotes fixed before and during 1st cytokinesis. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. Possible functions of this local disassembly include helping sister centrosomes move apart after mitosis, preparing a soft path for furrow ingression, and releasing G-actin from internal networks to build cortical networks that support furrow ingression.

Assembly of Spindles and Asters in Xenopus Egg Extracts.

Here, we provide methods for assembly of mitotic spindles and interphase asters in Xenopus laevis egg extract, and compare them to spindles and asters in the egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) extract and inducing entry into interphase extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes. CSF spindles are made by addition of sperm nuclei to CSF extract. They are less physiological and lack functional kinetochores but suffice for some applications. Large interphase asters are prepared by addition of artificial centrosomes or sperm nuclei to actin-intact egg extract. These asters grow rapidly to hundreds of microns in radius by branching microtubule nucleation at the periphery, so the aster as a whole is a network of short, dynamic microtubules. They resemble the sperm aster after fertilization, and the asters that grow out of the poles of the mitotic spindle at anaphase. When interphase asters grow into each other they interact and assemble aster interaction zones at their shared boundary. These zones consist of a line (in extract) or disc (in zygotes) of antiparallel microtubule bundles coated with cytokinesis midzone proteins. Interaction zones block interpenetration of microtubules from the two asters, and signal to the cortex to induce cleavage furrows. Their reconstitution in extract allows dissection of the biophysics of spatially regulated cytokinesis signaling.

Spindle-to-Cortex Communication in Cleaving Frog Eggs.

During cytokinesis, the mitotic spindle communicates with the cell cortex to position a cleavage furrow that will cut through the cell in the plane defined by the metaphase plate. We investigated the molecular basis of this communication in Xenopus laevis eggs, where the signal has to travel ∼400 µm in ∼30 min to reach the cortex from the first anaphase spindle. At anaphase onset, huge microtubule asters grow out from the poles of the spindle and meet at the plane previously defined by the metaphase plate. This disc-shaped boundary plane recruits the chromosome passenger complex (CPC) and centralspindlin to antiparallel microtubule bundles. It grows out to the cell cortex as the asters expand, where it induces the furrow. CPC and centralspindlin were not recruited to boundaries between asters from different spindles, suggesting a role of chromatin in triggering the CPC-positive state. Recruitment of CPC to aster boundaries was reconstituted in an extract system, and we observed that recruitment was stimulated by proximity to chromatin. Finally, we discuss models for molecular processes involved in initiation and growth of the CPC-positive disc that communicates the position of the metaphase plate to the cortex over hundreds of micrometers in frog eggs.

Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs.

Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.

Using supported bilayers to study the spatiotemporal organization of membrane-bound proteins.

Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.

Microtubule nucleation remote from centrosomes may explain how asters span large cells.

A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.

Spatial organization of cytokinesis signaling reconstituted in a cell-free system.

During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked the interpenetration of neighboring asters and recruited cytokinesis midzone proteins, including the chromosomal passenger complex (CPC) and centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed the recruitment of cleavage furrow markers, including an active RhoA reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.