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The role of cytoplasmic fragmentation in human embryo development and reproductive potential is widely recognized, albeit without standard definition nor agreed upon implication. While fragmentation is best understood to be a natural process across species, the origin of fragmentation remains incompletely understood and likely multifactorial. Several factors including embryo culture condition, gamete quality, aneuploidy, and abnormal cytokinesis seem to have important role in the etiology of cytoplasmic fragmentation. Fragmentation reduces the volume of cytoplasm and depletes embryo of essential organelles and regulatory proteins, compromising the developmental potential of the embryo. While it has been shown that degree of fragmentation and embryo implantation potential are inversely proportional, the degree, pattern, and distribution of fragmentation as it relates to pregnancy outcome is debated in the literature. This review highlights some of the challenges in analysis of fragmentation, while revealing trends in our evolving knowledge of how fragmentation may relate to functional development of the human embryos, implantation, and pregnancy outcome., (© 2024. The Author(s).)

Although abnormally fertilized zygotes with three or multiple pronuclei (3 PN/MPN) are commonly believed to be associated with improper maturation of the oocyte cytoplasm in conventional IVF cycles, no studies investigated the association between the proportion of MPN zygotes and the maturation state of the oocyte cohort. We compared the cytoplasmic maturity of oocytes from conventional IVF cycles with different proportions of 3 PN/MPN zygotes. A total of 1428 conventional IVF patients with ≥6 oocytes retrieved and fresh embryos transferred were divided into 4 groups according to the proportions of 3 PN/MPN zygotes. The pregnancy outcomes and the proportion of nuclear immature oocytes were analyzed to suggest the cytoplasmic maturation state of the oocyte cohort. Our results showed that the group with a low proportion of 3 PN/MPN zygotes had a higher clinical pregnancy rate (CPR) than those without 3 PN/MPN zygotes (P < 0.05). However, the live birth rate (LBR) was not significantly different between the two groups. The implantation rate (IR), CPR, and LBR did not differ between the low-proportion and high-proportion 3 PN/MPN groups. The proportion of nuclear immature oocytes on day 1 was highest in the group without 3 PN/MPN zygotes (23.8 %) and gradually decreased with an increased proportion of 3 PN/MPN zygotes (P < 0.001). Therefore, the presence of 3 PN/MPN zygotes after conventional IVF may indicate a more mature cytoplasmic state of the oocyte cohort, and the increased proportion of 3 PN/MPN zygotes is associated with an increased maturation state of the whole oocyte cohort. The occurrence and proportion of 3 PN/MPN zygotes may serve as an indicator for the cytoplasmic maturity of the oocyte cohort and help clinicians evaluate the efficiency of ovarian stimulation and optimize the stimulation protocols in subsequent cycles., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Many studies of cytoplasm rheology have focused on small components in the submicrometer scale. However, the cytoplasm also baths large organelles like nuclei, microtubule asters, or spindles that often take significant portions of cells and move across the cytoplasm to regulate cell division or polarization. Here, we translated passive components of sizes ranging from few up to ~50 percents of the cell diameter, through the vast cytoplasm of live sea urchin eggs, with calibrated magnetic forces. Creep and relaxation responses indicate that for objects larger than the micron size, the cytoplasm behaves as a Jeffreys material, viscoelastic at short timescales, and fluidizing at longer times. However, as component size approached that of cells, cytoplasm viscoelastic resistance increased in a nonmonotonic manner. Flow analysis and simulations suggest that this size-dependent viscoelasticity emerges from hydrodynamic interactions between the moving object and the static cell surface. This effect also yields to position-dependent viscoelasticity with objects initially closer to the cell surface being harder to displace. These findings suggest that the cytoplasm hydrodynamically couples large organelles to the cell surface to restrain their motion, with important implications for cell shape sensing and cellular organization.

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics, and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division, remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original positions. These forces are independent of cytoskeletal force generators yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization., Competing Interests: Competing interest statement: A patent on the magnetic method was deposited under the number PCT/EP2021/079072 on October 20, 2021., (Copyright © 2022 the Author(s). Published by PNAS.)

We show a double-functional fluorescence sensing paradigm that can retrieve nanometric pH information on biological structures. We use this method to measure the extent of protonic condensation around microtubules, which are protein polymers that play many roles crucial to cell function. While microtubules are believed to have a profound impact on the local cytoplasmic pH, this has been hard to show experimentally due to the limitations of conventional sensing techniques. We show that subtle changes in the local electrochemical surroundings cause a double-functional sensor to transform its spectrum, thus allowing a direct measurement of the protonic concentration at the microtubule surface. Microtubules concentrate protons by as much as one unit on the pH scale, indicating a charge storage role within the cell via the localized ionic condensation. These results confirm the bioelectrical significance of microtubules and reveal a sensing concept that can deliver localized biochemical information on intracellular structures.

Background: Information regarding the influence of cytoplasmic events during fertilisation on the clinical outcome remains limited. The cytoplasmic halo is one of these events. A previous study that used time-lapse technology found an association of the presence and morphokinetics of the cytoplasmic halo with cleavage patterns, development to the blastocyst stage, and the ongoing pregnancy rate after blastocyst transfer. Therefore, the cytoplasmic halo may be a useful predictor of the pregnancy outcome after cleaved embryo transfer. This study evaluated the ability of the cytoplasmic halo to predict a live birth after fresh cleaved embryo transfer on day 2, and sought to identify factors potentially influencing the presence and morphokinetics of the halo., Methods: A total of 902 embryos cultured in the EmbryoScope+® time-lapse system and subjected to single fresh cleaved embryo transfer were retrospectively analysed. The presence and duration of a cytoplasmic halo were annotated. The initial positions of the pronuclei were also observed. The correlation between the cytoplasmic halo and live birth was evaluated and the association of the cytoplasmic halo with patient, cycle, and embryonic characteristics was determined., Results: Absence of a cytoplasmic halo was associated with a significant decrease in the likelihood of a live birth after fresh cleaved embryo transfer. Prolongation of the halo, especially the duration of central repositioning of cytoplasmic granules, had an adverse impact on the live birth rate. The characteristics of the cytoplasmic halo were not affected by the ovarian stimulation method used, female age, the serum steroid hormone level on the day of trigger, or semen quality. However, the cytoplasmic halo was significantly affected by male age, oocyte diameter, and the initial position of the male pronucleus., Conclusions: Absence or prolongation of the cytoplasmic halo was negatively correlated with the live birth rate after fresh cleaved embryo transfer. The characteristics of the cytoplasmic halo were strongly associated with oocyte diameter, male age, and the initial position of the male pronucleus. These findings indicate that the characteristics of the cytoplasmic halo can be used to select more competent embryos for transfer at the cleavage stage., (© 2021. The Author(s).)

Living cells are a complex soft material with fascinating mechanical properties. A striking feature is that, regardless of their types or states, cells exhibit a universal power-law rheological behavior which to this date still has not been captured by a single theoretical model. Here, we propose a cellular structural model that accounts for the essential mechanical responses of cell membrane, cytoplasm and cytoskeleton. We demonstrate that this model can naturally reproduce the universal power-law characteristics of cell rheology, as well as how its power-law exponent is related to cellular stiffness. More importantly, the power-law exponent can be quantitatively tuned in the range of 0.1 ~ 0.5, as found in most types of cells, by varying the stiffness or architecture of the cytoskeleton. Based on the structural characteristics, we further develop a self-similar hierarchical model that can spontaneously capture the power-law characteristics of creep compliance over time and complex modulus over frequency. The present model suggests that mechanical responses of cells may depend primarily on their generic architectural mechanism, rather than specific molecular properties., (© 2021. The Author(s).)

Recent work has highlighted roles for thermodynamic phase behavior in diverse cellular processes. Proteins and nucleic acids can phase separate into three-dimensional liquid droplets in the cytoplasm and nucleus and the plasma membrane of animal cells appears tuned close to a two-dimensional liquid-liquid critical point. In some examples, cytoplasmic proteins aggregate at plasma membrane domains, forming structures such as the postsynaptic density and diverse signaling clusters. Here we examine the physics of these surface densities, employing minimal simulations of polymers prone to phase separation coupled to an Ising membrane surface in conjunction with a complementary Landau theory. We argue that these surface densities are a phase reminiscent of prewetting, in which a molecularly thin three-dimensional liquid forms on a usually solid surface. However, in surface densities the solid surface is replaced by a membrane with an independent propensity to phase separate. We show that proximity to criticality in the membrane dramatically increases the parameter regime in which a prewetting-like transition occurs, leading to a broad region where coexisting surface phases can form even when a bulk phase is unstable. Our simulations naturally exhibit three-surface phase coexistence even though both the membrane and the polymer bulk only display two-phase coexistence on their own. We argue that the physics of these surface densities may be shared with diverse functional structures seen in eukaryotic cells., Competing Interests: The authors declare no competing interest.

Nuclei import and export proteins, including cell cycle regulators. These import-export processes are modulated periodically by the cell cycle, for example due to the periodic assembly and breakdown of the nuclear envelope. As such, replicated DNA can be segregated between the two daughter cells and the proteins that were localized in the nucleus are free to diffuse throughout the cytoplasm. Here, we study a mathematical import-diffusion model to show how proteins, i.e. cell cycle regulators, could be redistributed in the cytoplasm by nuclei that periodically toggle between interphase and mitosis. We show that when the cell cycle period depends on the local concentration of regulators, the model exhibits mitotic waves. We discuss how the velocity and spatial origin of these mitotic waves depend on the different model parameters. This work is motivated by recent in vitro experiments reporting on mitotic waves in cycling cell-free extracts made with Xenopus laevis frog eggs, where multiple nuclei share the same cytoplasm. Such experiments have shown that nuclei act as pacemakers for the cell cycle and thus play an important role in collectively defining the spatial origin of mitotic waves., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The C. elegans germline is organized as a syncytium in which each germ cell possesses an intercellular bridge that is maintained by a stable actomyosin ring and connected to a common pool of cytoplasm, termed the rachis. How germ cells undergo cytokinesis while maintaining this syncytial architecture is not completely understood. Here, we use live imaging to characterize primordial germ cell (PGC) division in C. elegans first-stage larvae. We show that each PGC possesses a stable intercellular bridge that connects it to a common pool of cytoplasm, which we term the proto-rachis. We further show that the first PGC cytokinesis is incomplete and that the stabilized cytokinetic ring progressively moves towards the proto-rachis and eventually integrates with it. Our results support a model in which the initial expansion of the C. elegans syncytial germline occurs by incomplete cytokinesis, where one daughter germ cell inherits the actomyosin ring that was newly formed by stabilization of the cytokinetic ring, while the other inherits the pre-existing stable actomyosin ring. We propose that such a mechanism of iterative cytokinesis incompletion underpins C. elegans germline expansion and maintenance., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli 's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic., Competing Interests: The authors declare no competing interest.

Biomolecular condensates that form via phase separation are increasingly regarded as coordinators of cellular reactions that regulate a wide variety of biological phenomena. Mounting evidence suggests that multiple steps of the RNA life cycle are organized within RNA-binding protein-rich condensates. In this Review, we discuss recent insights into the influence of phase separation on RNA biology, which has implications for basic cell biology, the pathogenesis of human diseases and the development of novel therapies.

Apoptosis is closely associated with many diseases. Detection of apoptosis can be achieved by morphology, biochemistry, molecular biology, immunology, and other techniques. However, as technologies are increasingly used for the detection of apoptosis, many researchers are confused about how to choose a suitable method to detect apoptosis. Selection of a suitable detection method for apoptosis will help clinical diagnosis and prevention of diseases. This article reviews the selection of optimal apoptosis-detection methods based on research purposes and technique principles.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Key Message: Major fertility restorer locus for Aegilops kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS. Most likely candidate gene is TraesCS1B02G197400LC, which is predicted to encode a pectinesterase/pectinesterase inhibitor. Cytoplasmic male sterility (CMS) is widely used for heterosis and hybrid seed production in wheat. Genes related to male fertility restoration in the presence of Aegilops kotschyi cytoplasm have been reported, but the fertility restoration-associated gene loci have not been investigated systematically. In this study, a BC 1 F 1 population derived from a backcross between KTP116A, its maintainer line TP116B, and its restorer line LK783 was employed to map fertility restoration by bulked segregant RNA-Seq (BSR-Seq). A major fertility allele restorer locus for Ae. kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS, and it was contributed by LK783. Morphological and cytological studies showed that male fertility restoration occurred mainly after the late uninucleate stage. Based on simple sequence repeat and single-nucleotide polymorphism genotyping, the gene locus was located between Xnwafu&#95;6 and Xbarc137 on chromosome arm 1BS. To further isolate the specific region, six Kompetitive allele-specific polymerase chain reaction markers derived from BSR-Seq were developed to delimit Rfk1 within physical intervals of 26.0 Mb. After searching for differentially expressed genes within the candidate interval in the anthers and sequencing analysis, TraesCS1B02G197400LC was identified as a candidate gene for Rfk1 and it was predicted to encode a pectinesterase/pectinesterase inhibitor. Expression analysis also confirmed that it was specifically expressed in the anthers, and its expression level was higher in fertile lines compared with sterile lines. Thus, TraesCS1B02G197400LC was identified as the most likely candidate gene for Rfk1, thereby providing insights into the fertility restoration mechanism for K-type CMS in wheat.

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability-transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density-dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.

Research Question: Can artificial intelligence and advanced image analysis extract and harness novel information derived from cytoplasmic movements of the early human embryo to predict development to blastocyst?, Design: In a proof-of-principle study, 230 human preimplantation embryos were retrospectively assessed using an artificial neural network. After intracytoplasmic sperm injection, embryos underwent time-lapse monitoring for 44 h. For comparison, standard embryo assessment of each embryo by a single embryologist was carried out to predict development to blastocyst stage based on a single picture frame taken at 42 h of development. In the experimental approach, in embryos that developed to blastocyst or destined to arrest, cytoplasm movement velocity was recorded by time-lapse monitoring during the first 44 h of culture and analysed with a Particle Image Velocimetry algorithm to extract quantitative information. Three main artificial intelligence approaches, the k-Nearest Neighbour, the Long-Short Term Memory Neural Network and the hybrid ensemble classifier were used to classify the embryos., Results: Blind operator assessment classified each embryo in terms of ability to develop to blastocyst, with 75.4% accuracy, 76.5% sensitivity, 74.3% specificity, 74.3% precision and 75.4% F1 score. Integration of results from artificial intelligence models with the blind operator classification, resulted in 82.6% accuracy, 79.4% sensitivity, 85.7% specificity, 84.4% precision and 81.8% F1 score., Conclusions: The present study suggests the possibility of predicting human blastocyst development at early cleavage stages by detection of cytoplasm movement velocity and artificial intelligence analysis. This indicates the importance of the dynamics of the cytoplasm as a novel and valuable source of data to assess embryo viability., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration. IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly., (Copyright © 2021 Nair and Zlotnick.)

In arthropods, Wolbachia endosymbionts induce conditional sterility, called cytoplasmic incompatibility (CI), resulting from embryonic lethality. CI penetrance (i.e., embryonic death rate) varies depending on host species and Wolbachia strains involved. All Culex pipiens mosquitoes are infected by the endosymbiotic alphaproteobacteria Wolbachia w Pip. CI in Culex , characterized as a binary "compatible/incompatible" phenomenon, revealed an unparalleled diversity of patterns linked to the amplification-diversification of cidA and cidB genes. Here, we accurately studied CI penetrance variations in the light of cid genes divergence by generating a C. pipiens compatibility matrix between 11 lines hosting different phylogenetic w Pip groups and exhibiting distinct cid gene repertoires. We showed, as expected, that crosses involving w Pip from the same group were mostly compatible. In contrast, only 22% of the crosses involving different w Pip groups were compatible, while 54% were fully incompatible. For the remaining 24% of the crosses, "intermediate" compatibilities were reported, and a cytological observation of the first zygotic division confirmed the occurrence of "canonical" CI phenotypes in a fraction of the eggs. Backcross experiments demonstrated that intermediate compatibilities were not linked to host genetic background but to the Wolbachia strains involved. This previously unstudied intermediate penetrance CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants markedly divergent from other w Pip groups. Our data demonstrate that CI is not always a binary compatible/incompatible phenomenon in C. pipiens but that intermediate compatibilities putatively resulting from partial mismatch due to Cid proteins divergence exist in this species complex. IMPORTANCE Culex pipiens mosquitoes are infected with w Pip. These endosymbionts induce a conditional sterility called CI resulting from embryonic deaths, which constitutes a cornerstone for Wolbachia antivectorial methods. Recent studies revealed that (i) two genes, cidA and cidB , are central in Wolbachia -CI mechanisms, and (ii) compatibility versus incompatibility between mosquito lines depends on the w Pip phylogenetic groups at play. Here, we studied CI variations in relation to w Pip groups and cid genes divergence. We showed, as expected, that the crosses involving w Pip from the same group were compatible. In contrast, 78% of the crosses involving different w Pip groups were partially or fully incompatible. In such crosses, we reported defects during the first zygotic division, a hallmark of CI. We showed that CI was more severe and frequent in crosses involving w Pip-IV strains exhibiting cid variants, which markedly diverge from those of other w Pip groups., (Copyright © 2021 Sicard et al.)

Cortical actin waves have emerged as a widely prevalent phenomena and brought pattern formation to many fields of cell biology. Cortical excitabilities, reminiscent of the electric excitability in neurons, are likely fundamental property of the cell cortex. Although they have been mostly considered to be biochemical in nature, accumulating evidence support the role of mechanics in the pattern formation process. Both pattern formation and mechanobiology approach biological phenomena at the collective level, either by looking at the mesoscale dynamical behavior of molecular networks or by using collective physical properties to characterize biological systems. As such they are very different from the traditional reductionist, bottom-up view of biology, which brings new challenges and potential opportunities. In this essay, we aim to provide our perspectives on what the proposed mechanochemical feedbacks are and open questions regarding their role in cortical excitable and oscillatory dynamics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytoplasm is a gel-like crowded environment composed of various macromolecules, organelles, cytoskeletal networks, and cytosol. The structure of the cytoplasm is highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules are restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the crowded nature of the cytoplasm at the microscopic scale, large-scale reorganization of the cytoplasm is essential for important cellular functions, such as cell division and polarization. How such mesoscale reorganization of the cytoplasm is achieved, especially for large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, is only beginning to be understood. In this review, we will discuss recent advances in elucidating the molecular, cellular, and biophysical mechanisms by which the cytoskeleton drives cytoplasmic reorganization across different scales, structures, and species., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In contrast to the homeokinesis of mitosis, asymmetric division of cytoplasm is the conspicuous feature of meiosis in mammalian oocytes. Protein regulator of cytokinesis 1 (PRC1) is an important regulator during mitotic spindle assembly and cytoplasmic division, but its functions in oocyte meiosis and early embryo development have not been fully elucidated. In this study, we detected PRC1 expression and localization and revealed a nuclear, spindle midzone-related dynamic pattern throughout meiotic and mitotic progressions. Treatment of oocytes with the reagents taxol or nocodazole disturbed the distribution of PRC1 in metaphase II oocytes. Further, PRC1 depletion led to failure of first polar body (PB1) extrusion and spindle migration, aneuploidy and defective kinetochore-microtubule attachment and spindle assembly. Overexpression of PRC1 resulted in PB1 extrusion failure, aneuploidy and serious defects of spindle assembly. To investigate PRC1 function in early embryos, we injected Prc1 morpholino into zygotes and 2-cell stage embryos. Depletion of PRC1 in zygotes impaired 4-cell, morula and blastocyst formation. Loss of PRC1 in single or double blastomeres in 2-cell stage embryos significantly impaired cell division, indicating its indispensable role in early embryo development. Co-immunoprecipitation showed that PRC1 interacts with polo-like kinase 1 (PLK1), and functional knockdown and rescue experiments demonstrated that PRC1 recruits PLK1 to the spindle midzone to regulate cytoplasmic division during meiosis. Finally, kinesin family member 4 knockdown downregulates PRC1 expression and leads to PRC1 localization failure. Taken together, our data suggest PRC1 plays an important role during oocyte maturation and early embryonic development by regulating chromosome dynamics and cytoplasmic division., (© 2020 Federation of European Biochemical Societies.)

This work briefly surveys the diversity of selected subcellular characteristics in hyphal tip cells of the fungal kingdom (Mycota). Hyphae are filamentous cells that grow by tip extension. It is a highly polarised mechanism that requires a robust secretory system for the delivery of materials (e.g. membrane, proteins, cell wall materials) to sites of cell growth. These events result it the self-assembly of a Spitzenkörper (Spk), found most often in the Basidiomycota, Ascomycota, and Blastocladiomycota, or an apical vesicle crescent (AVC), present in the most Mucoromycota and Zoopagomycota. The Spk is a complex apical body composed of secretory vesicles, cytoskeletal elements, and signaling proteins. The AVC appears less complex, though little is known of its composition other than secretory vesicles. Both bodies influence hyphal growth and morphogenesis. Other factors such as cytoskeletal functions, endocytosis, cytoplasmic flow, and turgor pressure are also important in sustaining hyphal growth. Clarifying subcellular structures, functions, and behaviours through bioimagining analysis are providing a better understanding of the cell biology and phylogenetic relationships of fungi. LAY DESCRIPTION: Fungi are most familiar to the public as yeast, molds, and mushrooms. They are eukaryotic organisms that inhabit diverse ecological niches around the world and are critical to the health of ecosystems performing roles in decomposition of organic matter and nutrient recycling (Heath, 1990). Fungi are heterotrophs, unlike plants, and comprise the most successful and diverse phyla of eukaryotic microbes, interacting with all other forms of life in associations that range from beneficial (e.g., mycorrhizae) to antagonistic (e.g., pathogens). Some fungi can be parasitic or pathogenic on plants (e.g., Cryphonectria parasitica, Magnaporthe grisea), insects (e.g., Beauveria bassiana, Cordyceps sp.), invertebrates (e.g., Drechslerella anchonia), vertebrates (e.g., Coccidioides immitis, Candia albicans) and other fungi (e.g., Trichoderma viride, Ampelomyces quisqualis). The majority of fungi, however, are saprophytes, obtaining nutrition through the brake down of non-living organic matter., (© 2020 Royal Microscopical Society.)

Cytoplasmic incompatibility (CI) is the most common symbiont-induced reproductive manipulation. Specifically, symbiont-induced sperm modifications cause catastrophic mitotic defects in the fertilized embryo and ensuing lethality in crosses between symbiotic males and either aposymbiotic females or females harboring a different symbiont strain. However, if the female carries the same symbiont strain, then embryos develop properly, thereby imparting a relative fitness benefit to symbiont-transmitting mothers. Thus, CI drives maternally-transmitted bacteria to high frequencies in arthropods worldwide. In the past two decades, CI experienced a boom in interest due to its (i) deployment in worldwide efforts to curb mosquito-borne diseases, (ii) causation by bacteriophage genes, cifA and cifB , that modify sexual reproduction, and (iii) important impacts on arthropod speciation. This review serves as a gateway to experimental, conceptual, and quantitative themes of CI and outlines significant gaps in understanding CI's mechanism that are ripe for investigation from diverse subdisciplines in the life sciences., Competing Interests: JS, BL, SB No competing interests declared, (© 2020, Shropshire et al.)

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic., Competing Interests: The authors have declared that no competing interests exist.

Cell polarization is required to define body axes during development. The position of spatial cues for polarization is critical to direct the body axes. In Caenorhabditis elegans zygotes, the sperm-derived pronucleus/centrosome complex (SPCC) serves as the spatial cue to specify the anterior-posterior axis. Approximately 30 min after fertilization, the contractility of the cell cortex is relaxed near the SPCC, which is the earliest sign of polarization and called symmetry breaking (SB). It is unclear how the position of SPCC at SB is determined after fertilization. Here, we show that SPCC drifts dynamically through the cell-wide flow of the cytoplasm, called meiotic cytoplasmic streaming. This flow occasionally brings SPCC to the opposite side of the sperm entry site before SB. Our results demonstrate that cytoplasmic flow determines stochastically the position of the spatial cue of the body axis, even in an organism like C. elegans for which development is stereotyped.

The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.

In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is "compacted" or "supercompacted," and there are suggestions that the cytoplasm is "glass-like." Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA., (Copyright © 2020 Zhu et al.)

Yeast cells, when exposed to stress, can enter a protective state in which cell division, growth, and metabolism are down-regulated. They remain viable in this state until nutrients become available again. How cells enter this protective survival state and what happens at a cellular and subcellular level are largely unknown. In this study, we used electron tomography to investigate stress-induced ultrastructural changes in the cytoplasm of yeast cells. After ATP depletion, we observed significant cytosolic compaction and extensive cytoplasmic reorganization, as well as the emergence of distinct membrane-bound and membraneless organelles. Using correlative light and electron microscopy, we further demonstrated that one of these membraneless organelles was generated by the reversible polymerization of eukaryotic translation initiation factor 2B, an essential enzyme in the initiation of protein synthesis, into large bundles of filaments. The changes we observe are part of a stress-induced survival strategy, allowing yeast cells to save energy, protect proteins from degradation, and inhibit protein functionality by forming assemblies of proteins.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Intra-epidermal nerve endings, responsible for cutaneous perception of temperature, pain and itch, are conventionally described as passing freely between keratinocytes, from the basal to the granular layers of the epidermis. However, the recent discovery of keratinocyte contribution to cutaneous nociception implies that their anatomical relationships are much more intimate than what has been described so far. By studying human skin biopsies in confocal laser scanning microscopy, we show that intra-epidermal nerve endings are not only closely apposed to keratinocytes, but can also be enwrapped by keratinocyte cytoplasms over their entire circumference and thus progress within keratinocyte tunnels. As keratinocytes must activate intra-epidermal nerve endings to transduce nociceptive information, these findings may help understanding the interactions between the keratinocytes and nervous system. The discovery of these nerve portions progressing in keratinocyte tunnels is a strong argument to consider that contacts between epidermal keratinocytes and intra-epidermal nerve endings are not incidental and argue for the existence of specific and rapid paracrine communication from keratinocytes to sensory neurons., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown. C. elegans offers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretching during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular.

Many plant mutants are known that exhibit some degree of helical growth. This 'twisted' phenotype has arisen frequently in mutant screens of model organisms, but it is also found in cultivars of ornamental plants, including trees. The phenomenon, in many cases, is based on defects in cell expansion symmetry. Any complete model which explains the anisotropy of plant cell growth must ultimately explain how helical cell expansion comes into existence - and how it is normally avoided. While the mutations observed in model plants mainly point to the microtubule system, additional affected components involve cell wall functions, auxin transport and more. Evaluation of published data suggests a two-way mechanism underlying the helical growth phenomenon: there is, apparently, a microtubular component that determines handedness, but there is also an influence arising in the cell wall that feeds back into the cytoplasm and affects cellular handedness. This idea is supported by recent reports demonstrating the involvement of the cell wall integrity pathway. In addition, there is mounting evidence that calcium is an important relayer of signals relating to the symmetry of cell expansion. These concepts suggest experimental approaches to untangle the phenomenon of helical cell expansion in plant mutants., (© 2019 The Authors New Phytologist © 2019 New Phytologist Trust.)

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Intracellular thermometry has recently demonstrated temperatures in the nucleus, mitochondria, and centrosome to be significantly higher than those of the cytoplasm and cell membrane. This local thermogenesis and the resulting temperature gradient could facilitate the development of persistent, self-organizing convection currents in the cytoplasm of large eukaryotes. Using 3-dimensional computational simulations of intracellular fluid motion, we quantify the convective velocities that could result from the temperature differences observed experimentally. Based on these velocities, we identify the conditions necessary for this temperature-driven bulk flow to dominate over random thermal diffusive motion at the scale of a single eukaryotic cell. With temperature gradients of the order 1°C and diffusion coefficients comparable to those described in the literature, Péclet numbers ≥ 1 are feasible and permit comparable or greater effects of convection than diffusion in determining intracellular mass flux. In addition to the temperature gradient, the resulting flow patterns would also depend on the spatial localization of the heat source, the shape of the cell membrane, and the complex intracellular structure including the cytoskeleton. While this intracellular convection would be highly context-dependent, in certain settings, convective motion could provide a previously unrecognized mechanism for directed, bulk transport within eukaryotic cells., Competing Interests: The authors have declared that no competing interests exist.

Every daughter cell inherits two things from its mother: genetic information and a spatially organized complement of macromolecular complexes and organelles. The extent to which de novo self-organization, as opposed to inheritance of an already organized state, can suffice to yield functional cells is uncertain. We used Xenopus laevis egg extracts to show that homogenized interphase egg cytoplasm self-organizes over the course of ~30 minutes into compartments 300 to 400 micrometers in length that resemble cells. Formation of these cell-like compartments required adenosine triphosphate and microtubule polymerization but did not require added demembranated sperm nuclei with their accompanying centrosomes or actin polymerization. In cycling extracts with added sperm, the compartments underwent multiple cycles of division and reorganization, with mother compartments giving rise to two daughters at the end of each mitotic cycle. These results indicate that the cytoplasm can generate much of the spatial organization and cell cycle function of the early embryo., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli -like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA 4 , a kinase, and CheA 3 , a His-domain phosphorylated by CheA 4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY 6 . In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA 4 , while the other, TlpC, interacted with CheA 3 . Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell's metabolic state, generating the steady-state concentration of motor-stopping CheY 6 ~P. Membrane-cluster signalling produces a pulse of CheY 3 /CheY 4 ~P that displaces CheY 6 ~P and allows flagellar rotation and smooth swimming before both clusters adapt., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

While the nucleus-to-cytoplasmic (N:C) ratio has traditionally been used for assessing cell malignancy, most N:C measurement techniques are time-consuming and performed on thin histological sections, which prohibit assessment of three-dimensional cell structure. A combined ultrahigh frequency ultrasound (US) and photoacoustic (PA) technique was used to assess the size and N:C ratio of cultured cancer cells in three dimensions (3D). The diameters of the cells and their stained nuclei were obtained by fitting the power spectrum of backscattered US pulses and emitted PA waves, respectively, to well-established theoretical models. For comparison, an imaging flow cytometer (IFC) was also used to determine the two-dimensional cell and nucleus sizes from large cell populations using brightfield and fluorescence images, respectively. An N:C ratio was calculated for each cell using the quotient of the measured nucleus diameter and the total cell diameter. The mean N:C ratios calculated using the sound-based approach were 0.68, 0.66, and 0.54 for MCF-7, PC-3, and MDA-MB-231 cells, respectively, and were in good agreement with the corresponding values of 0.68, 0.67, and 0.68 obtained using the IFC. The combined US and PA technique, which assesses cellular N:C ratio in 3D, has potential applications in the detection of circulating tumor cells in liquid biopsies.

The intracellular dynamics of onion epidermal cells during the dehydration process is observed by holographic microscopy. Both the nucleus and cytoplasm are accurately revealed by quantitative phase imaging while dehydration takes place. Indeed, we notice that the contrast of phase images increases with the decrease in cellular water content. We foresee that such a dehydrating process can be effective for improving phase contrast, thus permitting better imaging of plant cells with the scope of learning more about cellular dynamics and related phenomena. Exploiting this concept, we observe intracellular cytoplasmic circulation and transport of biological material.

Phosphorus in plant cells occurs in inorganic form as both ortho- and pyrophosphate or bound to organic compounds, like e.g., nucleotides, phosphorylated metabolites, phospholipids, phosphorylated proteins, or phytate as P storage in the vacuoles of seeds. Individual compartments of the cell are surrounded by membranes that are selective barriers to avoid uncontrolled solute exchange. A controlled exchange of phosphate or phosphorylated metabolites is accomplished by specific phosphate transporters (PHTs) and the plastidial phosphate translocator family (PTs) of the inner envelope membrane. Plastids, in particular chloroplasts, are the site of various anabolic sequences of enzyme-catalyzed reactions. Apart from their role in metabolism PHTs and PTs are presumed to be also involved in communication between organelles and plant organs. Here we will focus on the integration of phosphate transport and homeostasis in signaling processes. Recent developments in this field will be critically assessed and potential future developments discussed. In particular, the occurrence of various plastid types in one organ (i.e. the leaf) with different functions with respect to metabolism or sensing, as has been documented recently following a tissue-specific proteomics approach (Beltran et al., 2018), will shed new light on functional aspects of phosphate homeostasis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hand-made cloning (HMC) is a method of choice for somatic cell nuclear transfer (SCNT). There is 20% to 50% of cytoplasm lost during manual enucleation of oocytes with HMC. To compensate, two enucleated demicytoplasts, instead of one, are fused with each donor cell, which leads to cytoplasm pooling from two different demicytoplasts. In this study, effects of using one, instead of two demicytoplasts (controls) was examined, for production of embryos using HMC. Use of one demicytoplast decreased blastocyst development (12.7 ± 1.98% compared with 47.6 ± 3.49%, P < 0.001), total cell number (TCN, 167.6 ± 14.66 compared with 335.9 ± 58.96, P < 0.01), apoptotic index (2.11 ± 0.38 compared with 3.43±0.38, P < 0.05) but did not significantly alter inner cell mass:trophectoderm cell number ratio (0.17 ± 0.01 compared with 0.19 ± 0.02) and the global content of H3K9ac and H3K27me3 of blastocysts, compared to controls. There were gene expression alterations in pluripotency- (SOX2 and NANOG but not OCT4), epigenetic- (DNMT1 but not DNMT3a and HDAC1), apoptosis- (CASPASE3 but not BCL-2 and BAX), trophectoderm- (CDX2), development- (G6PD but not GLUT1) and cell cycle check point control-related related genes (P53) compared with controls. Transfer of cloned blastocysts from one demicytoplast (n = 8) to recipients resulted in a live calf birth that after 12 days died whereas, with transfer of control blastocysts (n = 14) there was birth of a healthy calf. In conclusion, use of one, instead of two demicytoplasts for HMC, compromises in vitro developmental competence, and alters expression of several important genes affecting embryo development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease., (© 2019 New York Academy of Sciences.)

Maturation conditions and oocytes quality have substantial roles on developmental competence of unreconstructed or reconstructed oocytes. Cloning has been reported successfully with low efficiency through embryonic or somatic nuclear transfer into enucleated metaphase II oocytes. It has been suggested that introducing embryonic or somatic nucleus to cytoplast at earlier stage might improve reprogramming of the introduced nucleus. In addition, the synchronization between the donor nucleus and recipient cytoplasts might effect on reprogramming and further embryonic development. Therefore, the question arises; does the cytoplast of germinal vesicle (GV) oocyte containing nuclear sap improve developmental competence upon reconstruction with embryonic/somatic nucleus compared with MII cytoplast. It has been indicated that GV material is essential for remodeling of sperm or embryonic or somatic nucleus in GV oocyte cytoplast and their further embryonic developmental competence. GV cytoplasts could be obtained through micromanipulation. Different micromanipulation techniques of immature oocytes at different stages were adapted in addition to introducing donor nuclei at G0/G1, S and G2/M phase, and enucleolation technique as well. Upon micromanipulation, it could obtain GV cytoplasts; cumulus-free and without GV material, cumulus-complexes and without GV material, cumulus-free and with GV material, and cumulus-complexes and with GV material in addition to enucleolated GV oocytes. Therefore, this short review will address briefly the importance of maturation conditions, cumulus cells, oocyte quality, the techniques of enucleation GV oocyte, the cell cycle stage of the introduced donor cell, or nucleus for oocyte maturation and embryo development.

Background: Thermo-sensitive male-sterility based on Aegilops kotschyi cytoplasm (K-TCMS) plays an important role in hybrid wheat breeding. This has important possible applications in two-line hybrid wheat breeding but the genetic basis and molecular regulation mechanism related to fertility restoration are poorly understood. In this study, comparative transcriptome profiling based on RNA sequencing was conducted for two near-isogenic lines comprising KTM3315R and its sterile counterpart KTM3315A, a total of six samples (3 repetitions per group), in order to identify fertility restoration genes and their metabolic pathways., Results: In total, 2642 significant differentially expressed genes (DEGs) were detected, among which 1238 were down-regulated and 1404 were up-regulated in fertile anthers. Functional annotation enrichment analysis identified important pathways related to fertility restoration, such as carbohydrate metabolism, phenylpropanoid metabolism and biosynthesis, as well as candidate genes encoding pectin methylesterase and flavanone 3-hydroxylase. Moreover, transcription factor analysis showed that a large number of DEGs were mainly involved with the WRKY, bHLH, and MYB transcription factor families. Determination of total soluble sugar and flavonoid contents demonstrated that important metabolic pathways and candidate genes are associated with fertility restoration. Twelve DEGs were selected and detected by quantitative reverse-transcribed PCR, and the results indicated that the transcriptome sequencing results were reliable., Conclusions: Our results indicate that identified DEGs were related to the fertility restoration and they proved to be crucial in Aegilops kotschyi cytoplasm. These findings also provide a basis for exploring the molecular regulation mechanism associated with wheat fertility restoration as well as screening and cloning related genes.

Pasteurella multocida B:2 is a Gram-negative organism causing haemorrhagic septicaemia (HS) in buffaloes. It causes severe pulmonary infection, leading to infiltration of numerous macrophages and neutrophils. Despite the inflammatory response, buffaloes succumb to HS. This study aims to evaluate the in-vitro efficacy of macrophages and neutrophils of buffalo following exposure to P. multocida B:2. In-vitro infections were done using 10 7  cfu/ml of P. multocida B:2 for Group 1, Escherichia coli for Group 2 and Mannhaemia haemolytica A:2 for Group 3 cells. The inoculated cell cultures were harvested at 0, 30, 60 and 120 min post-exposure and the phagocytic, killing and cell death rates were determined. Both phagocytosis and killing rates of all bacteria increased over time. Phagocytosis involved between 71% and 73% neutrophils and between 60% and 64% macrophages at 120 min. Killing rate of all bacteria involved between 76% and 79% for neutrophils and between 70% and 74% for macrophages at 120 min. Death rate of neutrophils ranged between 67% in Group 3, and 88% in Group 1 at 120 min, significantly (p < 0.05) higher than Group 3 but insignificant (p > 0.05) than Group 2. Similar pattern was observed for death rate of macrophages. The phagocytosis and killing rates of P. multocida B:2 were similar to other bacterial species used in this study but more neutrophils and macrophages were dead following infection by P. multocida B:2 than M. haemolytica A:2., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation., (Copyright © 2019 Elsevier Inc. All rights reserved.)