Dynamics of hydraulic and contractile wave-mediated fluid transport during Drosophila oogenesis.

From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. Here, we combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. We discovered that during "nurse cell (NC) dumping" most cytoplasm is transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. Our work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila.

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. Although mechanisms have been established for individual cells' dynamic behaviors, the mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase activating protein (RhoGAP) pair required for actomyosin waves in egg chambers. Specifically, depletion of the RhoGEF, Ect2, or the RhoGAP, RhoGAP15B, disrupted actomyosin wave induction, and both proteins relocalized from the nucleus to the cortex preceding wave formation. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair, RhoGEF2 and Cumberland GAP (C-GAP), resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly by ∼4 s. We found that C-GAP was recruited to actomyosin waves, and disrupting F-actin polymerization altered the spatial organization of both RhoA signaling and the cytoskeleton in waves. In addition, disrupting F-actin dynamics increased wave period and width, consistent with a possible role for F-actin in promoting delayed negative feedback. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types, such as epithelial and syncytial cells.

Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.

Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila.

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. While mechanisms have been established for individual cells' dynamic behaviors, mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a RhoGEF and RhoGAP pair whose relocalization from nucleus to cortex results in actomyosin waves in egg chambers. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly and RhoGAP recruitment by ~4 seconds. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types.

Scaling behaviour and control of nuclear wrinkling.

The cell nucleus is enveloped by a complex membrane, whose wrinkling has been implicated in disease and cellular aging. The biophysical dynamics and spectral evolution of nuclear wrinkling during multicellular development remain poorly understood due to a lack of direct quantitative measurements. Here, we characterize the onset and dynamics of nuclear wrinkling during egg development in the fruit fly when nurse cell nuclei increase in size and display stereotypical wrinkling behavior. A spectral analysis of three-dimensional high-resolution live imaging data from several hundred nuclei reveals a robust asymptotic power-law scaling of angular fluctuations consistent with renormalization and scaling predictions from a nonlinear elastic shell model. We further demonstrate that nuclear wrinkling can be reversed through osmotic shock and suppressed by microtubule disruption, providing tuneable physical and biological control parameters for probing mechanical properties of the nuclear envelope. Our findings advance the biophysical understanding of nuclear membrane fluctuations during early multicellular development.

Vision impairment and refractive errors in refugees presenting to community optometry clinics in Victoria, Australia.

BACKGROUND: There is a paucity of data relating to refugee eye health in Australia. This study aimed at investigating the spectrum of vision impairment and other ocular conditions in refugees utilising the Victorian Eyecare Service operated by the Australian College of Optometry. METHODS: A cross-sectional study of electronic clinical records of 518 individuals (adults and children) recognised as refugees by the Australian College of Optometry and treated between January 2013 and May 2014 were identified. Extracted data included presenting visual acuities, best-corrected visual acuities, and final refraction values (using spherical equivalents), for both eyes. Diagnoses of presenting ocular conditions were also extracted. RESULTS: Of all refugees examined, 129 (27.2 per cent) had some degree of vision impairment (≤ 6/9.5) based on presenting visual acuities in their better eye; five (1.0 per cent) being of a severe (≤ 6/60) or profound (≤ 6/120) nature. In contrast, 27 (6.3 per cent) refugees had some degree of vision impairment based on best-corrected visual acuities in their better eye; two (0.4 per cent) being of a severe or profound nature. The prevalence of myopia (≥ -0.50 D) in the better eye was 23.0 per cent (n = 114); 25 (5.0 per cent) being moderate (≥ -3.00 D) to high (≥ -6.00 D). The prevalence of hypermetropia (≥ +2.00 D) in the better eye was 3.2 per cent (n = 16); 12 (2.4 per cent) being moderate (≥ +2.25 D) to high (≥ +5.25 D). The most common ocular conditions diagnosed at initial presentation were refractive error (n = 104, 20.1 per cent) and dry eyes (n = 57, 11.0 per cent). CONCLUSION: Mild vision impairment and refractive error are significant issues for refugees attending the Australian College of Optometry, emphasising the need for optometry, particularly refractive, services in this population.

Graphene-Sealed Flow Cells for In Situ Transmission Electron Microscopy of Liquid Samples.

We introduce a graphene-based nanofluidic cell that facilitates in situ imaging of liquid samples via transmission electron microscopy. The cell combines the benefits of graphene liquid cells-namely, high resolution, reduced charging effects, and excellent sample stability-with the ability to introduce reactants and control fluid concentrations as provided by conventional silicon-nitride-windowed flow cells. The graphene flow cell offers significantly less window bowing compared to existing commercial holders. We demonstrate the performance of the flow cell by imaging gold nanoparticle dynamics and uranyl acetate crystallization. Our results confirm the utility of graphene flow cells in obtaining the high spatial and temporal resolution required for probing the complex dynamics of nanoparticles and nucleation pathways in aqueous solutions.

Extended access to methamphetamine self-administration up-regulates dopamine transporter levels 72 hours after withdrawal in rats.

Previous studies have demonstrated that there are persistent changes in dopamine systems following withdrawal from methamphetamine (METH). This study examined changes in striatal dopamine transporter (DAT), tyrosine hydroxylase (TH) and dopamine receptor 2 (D2) 72 h after withdrawal from METH intravenous self- administration (IVSA). Rats were given limited (1h) or extended (6h) access to METH IVSA (0.05 mg/kg/0.1 ml infusion) for 22 days. Controls did not receive METH IVSA. The rats given extended access to IVSA displayed higher METH intake during the first hour of drug access compared to rats given limited access. Extended access to METH also produced a concomitant increase in striatal DAT levels relative to drug-naïve controls. There were no changes in TH or D2 levels across groups. Previous studies have reported a decrease in striatal DAT levels during protracted periods (>7 days) of withdrawal from METH IVSA. This study extends previous work by showing an increase in striatal DAT protein expression during an earlier time point of withdrawal from this drug. These results are an important step toward understanding the dynamic changes in dopamine systems that occur during different time points of withdrawal from METH IVSA.

Evidence for association of endothelial nitric oxide synthase gene in subjects with glaucoma and a history of migraine.

PURPOSE: There is evidence to suggest that vasospasm and vascular dysregulation play a role in the etiology of glaucoma. This effect may be particularly relevant in patients with glaucoma who have a history of migraine or vasospastic tendencies. This study was conducted to investigate the role of genes with products that regulate blood flow to ocular tissues. The candidate genes were the two isoforms of nitric oxide synthase (NOS), NOS3 and -2A, and endothelin (ET)-l. The frequency of the T786C mutation in NOS3 was also examined. METHODS: DNA was obtained from 58 patients with primary open-angle glaucoma (POAG), 76 with normal tension glaucoma (NTG), and 38 control subjects. Polymerase chain reactions (PCR) were used to compare the frequency of the alleles between the subjects with glaucoma and the control subjects and the subjects with glaucoma with vasospasm or migraine. The PCR product was sequenced to identify the frequency of the T786C mutation. RESULTS: The distribution of the NOS3 repeat alleles in subjects with glaucoma and control subjects just failed to reach statistical significance (P = 0.06). The distribution in subjects with NTG or POAG did not differ significantly. A significant difference was found (P < 0.001) in the distribution of allele frequencies of the NOS3 marker in subjects who had glaucoma with migraine versus control subjects. There were no differences in the distribution of the NOS2A or ET-1 markers between the subjects with glaucoma and the control subjects. CONCLUSIONS: This study provides evidence of an association between the NOS3 gene and subjects with glaucoma who have a history of migraine. Unlike in other studies, no evidence was found of an association between ET-1 and glaucoma.

Effects of dopamine D(2)-like receptor agonists in mice trained to discriminate cocaine from saline: influence of feeding condition.

In rats, the discriminative stimulus effects of direct- and indirect-acting dopamine receptor agonists are mediated by multiple dopamine receptor subtypes and the relative contribution of dopamine D2 and D3 receptors to these effects varies as a function of feeding condition. In these studies, free-fed and food-restricted mice were trained to discriminate 10.0mg/kg cocaine using a two-lever discrimination procedure in which responding was maintained by food. Both groups of mice acquired the discrimination; however, free-fed mice responded at lower rates than food-restricted mice. Dopamine D3 receptor agonists, pramipexole and quinpirole, increased cocaine-appropriate responding (>85%) in food-restricted, but not in free-fed mice. The dopamine D2 receptor agonist, sumanirole, and the nonselective dopamine receptor agonist, apomorphine, failed to increase cocaine-appropriate responding in either group. Free-fed mice were more sensitive than food-restricted mice to the rate-decreasing effects of dopamine receptor agonists and these effects could not be overcome by increasing the magnitude of reinforcement. Because feeding condition did not alter quinpirole-induced hypothermia, it is unlikely that differences in the discriminative stimulus or rate-decreasing effects of dopamine D2-like receptor agonists were due to differences in the pharmacokinetic properties of the drugs. Although these results suggest that the discriminative stimulus effects of cocaine are mediated by both dopamine D2 and D3 receptors in food-restricted mice, the increased sensitivity of free-fed mice to the rate-decreasing effects of dopamine D2-like receptor agonists limited conclusions about the impact of feeding conditions on the relative contribution of dopamine D2 and D3 receptors to the discriminative stimulus effects of cocaine.