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Microscopy core facilities are increasingly utilised research resources, but they are generally only available to users within the host institution. Such localised access misses an opportunity to facilitate research across a broader user base. Here, we present the model of an open-access microscopy facility, using the Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus as an example. The AIC has pioneered a model whereby advanced microscopy technologies and expertise are made accessible to researchers on a global scale. We detail our experiences in addressing the considerable challenges associated with this model for those who may be interested in launching an open-access imaging facility. Importantly, we focus on how this model can empower researchers, particularly those from resource-constrained settings., (© 2023 Royal Microscopical Society.)

Rift Valley fever virus (RVFV) is a hemorrhagic fever virus with the potential for significant economic and public health impact. Vaccination with an attenuated strain, DelNSsRVFV, provides protection from an otherwise lethal RVFV challenge, but mechanistic determinants of protection are undefined. In this study, a murine model was used to assess the contributions of humoral and cellular immunity to DelNSsRVFV-mediated protection. Vaccinated mice depleted of T cells were protected against subsequent challenge, and passive transfer of immune serum from vaccinated animals to naïve animals was also protective, demonstrating that T cells were dispensable in the presence of humoral immunity and that humoral immunity alone was sufficient. Animals depleted of B cells and then vaccinated were protected against challenge. Total splenocytes, but not T cells alone, B cells alone, or B + T cells harvested from vaccinated animals and then transferred to naïve animals were sufficient to confer protection, suggesting that multiple cellular interactions were required for effective cellular immunity. Together, these data indicate that humoral immunity is sufficient to confer vaccine-mediated protection and suggests that cellular immunity plays a role in protection that requires the interaction of various cellular components., (© 2022. The Author(s).)

Nitrate (NO 3 - ) uptake and distribution are critical to plant life. Although the upstream regulation of NO 3 - uptake and downstream responses to NO 3 - in a variety of cells have been well studied, it is still not possible to directly visualize the spatial and temporal distribution of NO 3 - with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named NitraMeter3.0, for the quantitative visualization of NO 3 - distribution in Arabidopsis thaliana . This biosensor tracked the spatiotemporal distribution of NO 3 - along the primary root axis and disruptions by genetic mutation of transport (low NO 3 - uptake) and assimilation (high NO 3 - accumulation). The developed biosensor effectively monitors NO 3 - concentrations at the cellular level in real time and spatiotemporal changes during the plant life cycle.

Rift Valley fever (RVF) is an arboviral disease of humans and livestock responsible for severe economic and human health impacts. In humans, RVF spans a variety of clinical manifestations, ranging from an acute flu-like illness to severe forms of disease, including late-onset encephalitis. The large variations in human RVF disease are inadequately represented by current murine models, which overwhelmingly die of early-onset hepatitis. Existing mouse models of RVF encephalitis are either immunosuppressed, display an inconsistent phenotype, or develop encephalitis only when challenged via intranasal or aerosol exposure. In this study, the genetically defined recombinant inbred mouse resource known as the Collaborative Cross (CC) was used to identify mice with additional RVF disease phenotypes when challenged via a peripheral foot-pad route to mimic mosquito-bite exposure. Wild-type Rift Valley fever virus (RVFV) challenge of 20 CC strains revealed three distinct disease phenotypes: early-onset hepatitis, mixed phenotype, and late-onset encephalitis. Strain CC057/Unc, with the most divergent phenotype, which died of late-onset encephalitis at a median of 11 days post-infection, is the first mouse strain to develop consistent encephalitis following peripheral challenge. CC057/Unc mice were directly compared to C57BL/6 mice, which uniformly succumb to hepatitis within 2-4 days of infection. Encephalitic disease in CC057/Unc mice was characterized by high viral RNA loads in brain tissue, accompanied by clearance of viral RNA from the periphery, low ALT levels, lymphopenia, and neutrophilia. In contrast, C57BL/6 mice succumbed from hepatitis at 3 days post-infection with high viral RNA loads in the liver, viremia, high ALT levels, lymphopenia, and thrombocytopenia. The identification of a strain of CC mice as an RVFV encephalitis model will allow for future investigation into the pathogenesis and treatment of RVF encephalitic disease and indicates that genetic background makes a major contribution to RVF disease variation., Competing Interests: The authors have declared that no competing interests exist.

Biomolecular condensates formed via liquid-liquid phase separation enable spatial and temporal organization of enzyme activity. Phase separation in many eukaryotic condensates has been shown to be responsive to intracellular adenosine triphosphate (ATP) levels, although the consequences of these mechanisms for enzymes sequestered within the condensates are unknown. Here, we show that ATP depletion promotes phase separation in bacterial condensates composed of intrinsically disordered proteins. Enhanced phase separation promotes the sequestration and activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune enzyme sequestration and reactivity in response to the metabolic state of bacterial cells.

Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Rift Valley fever virus (RVFV) is an arbovirus found throughout Africa. It causes disease that is typically mild and self-limiting; however, some infected individuals experience severe manifestations, including hepatitis, encephalitis, or even death. Reports of RVFV encephalitis are notable among immunosuppressed individuals, suggesting a role for adaptive immunity in preventing this severe complication. This phenomenon has been modeled in C57BL/6 mice depleted of CD4 T cells prior to infection with DelNSs RVFV (RVFV containing a deletion of nonstructural protein NSs), resulting in late-onset encephalitis accompanied by high levels of viral RNA in the brain in 30% of animals. In this study, we sought to define the specific type(s) of CD4 T cells that mediate protection from RVFV encephalitis. The viral epitopes targeted by CD4 and CD8 T cells were defined in C57BL/6 mice, and tetramers for both CD4 and CD8 T cells were generated. RVFV-specific CD8 T cells were expanded and of a cytotoxic and proliferating phenotype in the liver following infection. RVFV-specific CD4 T cells were identified in the liver and spleen following infection and phenotyped as largely Th1 or Tfh subtypes. Knockout mice lacking various aspects of pathways important in Th1 and Tfh development and function were used to demonstrate that T-bet, CD40, CD40L, and major histocompatibility complex class II (MHC-II) mediated protection from RVFV encephalitis, while gamma interferon (IFN-γ) and interleukin-12 (IL-12) were dispensable. Virus-specific antibody responses correlated with protection from encephalitis in all mouse strains, suggesting that Tfh/B cell interactions modulate clinical outcome in this model. IMPORTANCE The prevention of RVFV encephalitis requires intact adaptive immunity. In this study, we developed reagents to detect RVFV-specific T cells and provide evidence for Tfh cells and CD40/CD40L interactions as critical mediators of this protection.

Discovered in 1931, Rift Valley fever virus (RVFV) is an arbovirus that causes disease in humans and livestock. In humans, disease ranges from a self-limiting febrile illness to a more severe hepatitis or encephalitis. There are currently no licensed human therapeutics for RVFV disease. Given the recent advances in the use of monoclonal antibodies (MAbs) for treating infectious disease, a panel of anti-RVFV Gn glycoprotein MAbs was developed and characterized. RVFV MAbs spanned a range of neutralizing abilities and mapped to distinct epitopes along Gn. The contribution of Fc effector functions in providing MAb-mediated protection from RVFV was assessed. IgG2a version MAbs had increased capacity to induce effector functions and conferred better protection from RVFV challenge in a lethal mouse model than IgG1 version MAbs. Overall, this study shows that Fc-mediated functions are a critical component of humoral protection from RVFV. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-borne virus found throughout Africa and into the Middle East. It has a substantial disease burden; in areas of endemicity, up to 60% of adults are seropositive. With a case fatality rate of up to 3% and the ability to cause hemorrhagic fever and encephalitis, RVFV poses a serious threat to human health. Despite the known human disease burden and the fact that it is a NIAID category A priority pathogen and a WHO priority disease for research and development, there are no vaccines or therapeutics available for RVF. In this study, we developed and characterized a panel of monoclonal antibodies against the RVFV surface glycoprotein, Gn. We then demonstrated therapeutic efficacy in the prevention of RVF in vivo in an otherwise lethal mouse model. Finally, we revealed a role for Fc-mediated function in augmenting the protection provided by these antibodies.

Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca 2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca 2+ indicator, MatryoshCaMP6s.

Receptor Tyrosine Kinases (RTKs) comprise a diverse group of cell-surface receptors that mediate key signaling events during animal development and are frequently activated in cancer. We show here that deletion of the extracellular regions of 10 RTKs representing 7 RTK classes or their substitution with the dimeric immunoglobulin Fc region results in constitutive receptor phosphorylation but fails to result in phosphorylation of downstream signaling effectors Erk or Akt. Conversely, substitution of RTK extracellular regions with the extracellular region of the Epidermal Growth Factor Receptor (EGFR) results in increases in effector phosphorylation in response to EGF. These results indicate that the activation signal generated by the EGFR extracellular region is capable of activating at least seven different RTK classes. Failure of phosphorylated Fc-RTK chimeras or RTKs with deleted extracellular regions to stimulate phosphorylation of downstream effectors indicates that either dimerization and receptor phosphorylation per se are insufficient to activate signaling or constitutive dimerization leads to pathway inhibition., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Rift Valley fever virus (RVFV) is a zoonotic arbovirus affecting humans and livestock in Africa and the Arabian Peninsula. The majority of human cases are mild and self-limiting; however, severe cases can result in hepatitis, encephalitis, or hemorrhagic fever. There is a lack of immunocompetent mouse models that faithfully recapitulate the varied clinical outcomes of RVF in humans. However, there are easily accessible and commonly used inbred mouse strains that have never been challenged with wild-type RVFV. Here, RVFV susceptibility and pathogenesis were evaluated across five commonly used inbred laboratory mouse strains: C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, A/J, and NZO/HILtJ. Comparisons between different mouse strains, challenge doses, and sexes revealed exquisite susceptibility to wild-type RVFV in an almost uniform manner. Never before challenged NOD/ShiLtJ, A/J, and NZO/HILtJ mice showed similar phenotypes of Rift Valley fever disease as previously tested inbred mouse strains. The majority of infected mice died or were euthanized by day 5 post-infection due to overwhelming hepatic disease as evidenced by gross liver pathology and high viral RNA loads in the liver. Mice surviving past day 6 across all strains succumbed to late-onset encephalitis. Remarkably, sex was not found to impact survival or viral load and showed only modest effect on time to death and weight loss for any of the challenged mouse strains following RVFV infection. Regardless of sex, these inbred mouse strains displayed extreme susceptibility to wild-type RVFV down to one virus particle., (Copyright © 2020 Cartwright, Barbeau and McElroy.)

Coral reefs, and their associated diverse ecosystems, are of enormous ecological importance. In recent years, coral health has been severely impacted by environmental stressors brought on by human activity and climate change, threatening the extinction of several major reef ecosystems. Reef damage is mediated by a process called 'coral bleaching' where corals, sea anemones, and other cnidarians lose their photosynthetic algal symbionts (family Symbiodiniaceae) upon stress induction, resulting in drastically decreased host energy harvest and, ultimately, coral death. The mechanism by which this critical cnidarian-algal symbiosis is lost remains poorly understood. The larvae of the sea anemone, Exaiptasia pallida (commonly referred to as 'Aiptasia') are an attractive model organism to study this process, but they are large (∼100 mm in length, ∼75 mm in diameter), deformable, and highly motile, complicating long-term imaging and limiting study of this critical endosymbiotic relationship in live organisms. Here, we report 'Traptasia', a simple microfluidic device with multiple traps designed to isolate and image individual, live larvae of Aiptasia and their algal symbionts over extended time courses. Using a trap design parameterized via fluid flow simulations and polymer bead loading tests, we trapped Aiptasia larvae containing algal symbionts and demonstrated stable imaging for >10 hours. We visualized algae within Aiptasia larvae and observed algal expulsion under an environmental stressor. To our knowledge, this device is the first to enable time-lapsed, high-throughput live imaging of cnidarian larvae and their algal symbionts and, in further implementation, could provide important insights into the cellular mechanisms of cnidarian bleaching under different environmental stressors. The 'Traptasia' device is simple to use, requires minimal external equipment and no specialized training to operate, and can easily be adapted using the trap optimization data presented here to study a variety of large, motile organisms.

Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Approximately 30%-40% of global CO 2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO 2 -fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls., (Copyright © 2015, American Association for the Advancement of Science.)

Pre-mitotic establishment of polarity is a key event in the preparation of mother cells for asymmetric cell divisions that produce daughters of distinct fates, and ensures correct cellular patterning of tissues and eventual organ function. Previous work has shown that two receptor-like kinases, PANGLOSS2 (PAN2) and PAN1, and the small GTPase RHO GTPASE OF PLANTS (ROP) promote mother cell polarity and subsequent division asymmetry in developing maize stomata. PAN proteins become polarized prior to asymmetric cell division, however, the mechanism of this polarization is unknown. Here we show that the SCAR/WAVE regulatory complex, which activates the actin-nucleating ARP2/3 complex, is the first known marker of polarity in this asymmetric division model and is required for PAN polarization. These findings implicate actin, and specifically branched actin networks, in PAN polarization and asymmetric cell division.

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days. We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging(1) (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers(2). This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution. Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors(3).

Myoblast fusion in the Drosophila embryos is a complex process that includes changes in cell movement, morphology and behavior over time. The advent of fluorescent proteins (FPs) has made it possible to track and image live cells, to capture the process of myoblast fusion, and to carry out quantitative analysis of myoblasts in real time. By tagging proteins with FPs, it is also possible to monitor the subcellular events that accompany the fusion process. Herein, we discuss the recent progress that has been made in imaging myoblast fusion in Drosophila, reagents that are now available, and microscopy conditions to consider. Using an Actin-FP fusion protein along with a membrane marker to outline the cells, we show the dynamic formation and breakdown of F-actin foci at sites of fusion. We also describe the methods used successfully to show that these foci are primarily if not wholly present in the fusion-competent myoblasts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion.

For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Polarization of cell division is essential for eukaryotic development, but little is known about how this is accomplished in plants. The formation of stomatal complexes in maize involves the polarization of asymmetric subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC), apparently under the influence of a GMC-derived signal. We found that the maize pan1 gene promotes the premitotic polarization of SMCs and encodes a leucine-rich repeat receptor-like protein that becomes localized in SMCs at sites of GMC contact. PAN1 has an inactive kinase domain but is required for the accumulation of a membrane-associated phosphoprotein, suggesting a function for PAN1 in signal transduction. Our findings implicate PAN1 in the transmission of an extrinsic signal that polarizes asymmetric SMC divisions toward GMCs.

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 and Brk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates the Brk pathway in local regulation of actin polymerization in plant cells.

Four maize (Zea mays L.) varieties were examined for ultraviolet radiation-induced changes in leaf rolling, biomass, fluctuating leaf asymmetry and DNA damage. Short-term dose-response curves for each response were constructed and responses in each line compared. The four varieties each exhibited a different pattern of tolerance and reactivity, ranging from B73, which was tolerant in all four measures, to TS1, which was affected in DNA damage levels and leaf rolling but unaffected in biomass accumulation and fluctuating leaf asymmetry. The pattern of ultraviolet radiation responses allows us to narrow the possibilities for the source of the defect in reactive varieties. The four varieties tested include inbred parents that have been used to construct recombinant inbred lines and a variety that is found in the background of the engineered RescueMu transposon mutagenesis lines. These dose-response curves and variety comparisons provide the foundation for genetic dissection of the mechanisms of ultraviolet radiation responses in maize.