Envelope Assembly Research Articles

Loss of YhcB results in overactive fatty acid biosynthesis.

Loss of the Escherichia coli inner membrane protein YhcB results in pleomorphic cell morphology and clear growth defects. Prior work suggested that YhcB was directly involved in cell division or peptidoglycan assembly. We found that loss of YhcB is detrimental in genetic backgrounds in which lipopolysaccharide (LPS) or glycerophospholipid (GPL) synthesis is altered. The growth defect of ΔyhcB could be rescued through inactivation of the Mla pathway, a system responsible for the retrograde transport of GPLs that are mislocalized to the outer leaflet of the outer membrane. Interestingly, this rescue was dependent upon the outer membrane phospholipase PldA that cleaves GPLs at the bacterial surface. Since the freed fatty acids resulting from PldA activity serve as a signal to the cell to increase LPS synthesis, this result suggested that outer membrane lipids are imbalanced in ΔyhcB. Mutations that arose in ΔyhcB populations during two independent suppressor screens were in genes encoding subunits of the acetyl coenzyme A carboxylase complex, which initiates fatty acid biosynthesis (FAB). These mutations fully restored cell morphology and reduced GPL levels, which were increased compared to wild-type bacteria. Growth of ΔyhcB with the FAB-targeting antibiotic cerulenin also increased cellular fitness. Furthermore, genetic manipulation of FAB and lipid biosynthesis showed that decreasing FAB rescued ΔyhcB filamentation, whereas increasing LPS alone could not. Altogether, these results suggest that YhcB may play a pivotal role in regulating FAB and, in turn, impact cell envelope assembly and cell division.IMPORTANCESynthesis of the Gram-negative cell envelope is a dynamic and complex process that entails careful coordination of many biosynthetic pathways. The inner and outer membranes are composed of molecules that are energy intensive to synthesize, and, accordingly, these synthetic pathways are under tight regulation. The robust nature of the Gram-negative outer membrane renders it naturally impermeable to many antibiotics and therefore a target of interest for antimicrobial design. Our data indicate that when the inner membrane protein YhcB is absent in Escherichia coli, the pathway for generating fatty acid substrates needed for all membrane lipid synthesis is dysregulated which leads to increased membrane material. These findings suggest a potentially novel regulatory mechanism for controlling the rate of fatty acid biosynthesis.

Nuclear envelope assembly relies on CHMP-7 in the absence of BAF-LEM-mediated hole closure.

Barrier-to-autointegration factor (BAF) protein is a DNA-binding protein that crosslinks chromatin to allow mitotic nuclear envelope (NE) assembly. The LAP2-emerin-MAN1 (LEM)-domain protein LEMD2 and ESCRT-II/III hybrid protein CHMP7 close NE holes surrounding spindle microtubules (MTs). BAF binds LEM-domain family proteins to repair NE ruptures in interphase, but whether BAF-LEM binding participates in NE hole closure around spindle MTs is not known. Here, we took advantage of the stereotypical event of NE formation in fertilized Caenorhabditis elegans oocytes to show that BAF-LEM binding and LEM-2-CHMP-7 have distinct roles in NE closure around spindle MTs. LEM-2 and EMR-1 (homologs of LEMD2 and emerin) function redundantly with BAF-1 (the C. elegans BAF protein) in NE closure. Compromising BAF-LEM binding revealed an additional role for EMR-1 in the maintenance of the NE permeability barrier. In the absence of BAF-LEM binding, LEM-2-CHMP-7 was required for NE assembly and embryo survival. The winged helix domain of LEM-2 recruits CHMP-7 to the NE in C. elegans and a LEM-2-independent nucleoplasmic pool of CHMP-7 also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis.

First person – Sarah Barger

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Sarah Barger is first author on ‘ Nuclear envelope assembly relies on CHMP-7 in the absence of BAF–LEM-mediated hole closure’, published in JCS. Sarah is a Postdoctoral Fellow in the lab of Shirin Bahmanyar at Yale University, USA, investigating the role of nuclear envelope proteins in cancer initiation and progression.

RCC1 functions as a tumor facilitator in clear cell renal cell carcinoma by dysregulating cell cycle, apoptosis, and EZH2 stability.

RCC1 functions as a pivotal guanine nucleotide exchange factor and was reported to be involved in mitosis, the assembly of the nuclear envelope, nucleocytoplasmic transport in cell physiological processes. Recent studies reported that RCC1 could regulate immunological pathways and promote the growth of some malignant solid tumors. However, the prognostic value and exact function of RCC1 remain unknown in patients with clear cell renal cell carcinoma (cRCC). The UALCAN and KM plotter portals were used to analyze the expression profile and related tumor prognosis of RCC1 in ccRCC using data from TCGA. The expression profile of RCC1 was also confirmed in clinical samples using qRT-PCR, western blotting, and immunohistochemistry. The role of RCC1 on ccRCC cells in vitro was confirmed by a series of functional assays. Animal experiments were performed to verify the suppressive effect of RCC1 knockdown on tumor growth in vivo. The correlation of RCC1 expression with that of EZH2 was explored in clinical samples using IHC. The interaction between RCC1 and EZH2 was further verified using a CO-IP assay and a protein stability assay. RCC1 was upregulated in ccRCC tissues compared with normal tissues in TCGA dataset and paired clinical samples. RCC1 promoted ccRCC progression by accelerating the cell cycle and suppressing apoptosis. In addition, RCC1 could bind EZH2 and regulate its expression at the posttranscriptional level. RCC1 and EZH2 expression showed a strong correlation in clinical samples. Further investigation proved that RCC1 regulated EZH2 protein stability through the ubiquitin-proteasome pathway. RCC1 could be a potential therapeutic target in ccRCC. The RCC1/EZH2 axis takes part in the development of ccRCC.

A tubule-sheet continuum model for the mechanism of nuclear envelope assembly.

Nuclear envelope (NE) assembly defects cause chromosome fragmentation, cancer, and aging. However, major questions about the mechanism of NE assembly and its relationship to nuclear pathology are unresolved. In particular, how cells efficiently assemble the NE starting from vastly different, cell type-specific endoplasmic reticulum (ER) morphologies is unclear. Here, we identify a NE assembly mechanism, "membrane infiltration," that defines one end of a continuum with another NE assembly mechanism, "lateral sheet expansion," in human cells. Membrane infiltration involves the recruitment of ER tubules or small sheets to the chromatin surface by mitotic actin filaments. Lateral sheet expansion involves actin-independent envelopment of peripheral chromatin by large ER sheets that then extend over chromatin within the spindle. We propose a "tubule-sheet continuum" model that explains the efficient NE assembly from any starting ER morphology, the cell type-specific patterns of nuclear pore complex (NPC) assembly, and the obligatory NPC assembly defect of micronuclei.

Coordination of bacterial cell wall and outer membrane biosynthesis

Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.

Azido Inositol Probes Enable Metabolic Labeling of Inositol-Containing Glycans and Reveal an Inositol Importer in Mycobacteria.

Bacteria from the genus Mycobacterium include pathogens that cause serious diseases in humans and remain as difficult infectious agents to treat. Central to these challenges are the composition and organization of the mycobacterial cell envelope, which includes unique and complex glycans. Inositol is an essential metabolite for mycobacteria due to its presence in the structural core of the immunomodulatory cell envelope glycolipids phosphatidylinositol mannoside (PIM) and PIM-anchored lipomannan (LM) and lipoarabinomannan (LAM). Despite their importance to mycobacterial physiology and pathogenesis, many aspects of PIM, LM, and LAM construction and dynamics remain poorly understood. Recently, probes that allow metabolic labeling and detection of specific mycobacterial glycans have been developed to investigate cell envelope assembly and dynamics. However, these tools have been limited to peptidoglycan, arabinogalactan, and mycolic acid-containing glycolipids. Herein, we report the development of synthetic azido inositol (InoAz) analogues as probes that can metabolically label PIMs, LM, and LAM in intact mycobacteria. Additionally, we leverage an InoAz probe to discover an inositol importer and catabolic pathway in Mycobacterium smegmatis. We anticipate that in the future, InoAz probes, in combination with bioorthogonal chemistry, will provide a valuable tool for investigating PIM, LM, and LAM biosynthesis, transport, and dynamics in diverse mycobacterial organisms.

Effects of LytR on bacterial morphology in Streptococcus pyognes

A robust cell envelope is the first line of defense in infectious pathogens when encountering host immune defenses. In Gram-positive bacteria, LytR-CpsA-Psr (LCP) family proteins play a major role in the synthesis and assembly of the cell envelope. The Gram-positive bacterium S. pyogenes causes a wide range of diseases from pharyngitis to septicemia, but the involvement of LytR in the synthesis and assembly of the cell wall envelope in this bacterium is not clear. Therefore, we investigated whether LytR of S. pyogenes, like LytR of other streptococci, is involved in cell wall formation. The lytR gene-deficient strains were used to investigate the bacteria's ability to form a chaining, drug sensitivity to penicillin G, an inhibitor of cell wall synthesis, bacterial biofilm formation, and the morphological structure of bacteria by transmission electron microscopy, and the expression of pbp2b, which encodes a penicillin-binding protein, compared with the wild-type strain. Our results showed that LytR-deficient strains had reduced bacterial chaining compared to wild-type strains. Compared to wild-type strains, LytR-deficient strains were also more drug sensitive to the cell wall synthesis inhibitor penicillin G. This genetic study was accompanied by increased expression of the pbp2b gene in the LytR-deficient strains. In addition, the LytR-deficient strain showed a reduced ability to form biofilms, and the lytR gene-deficient strain showed morphological irregularities with abnormal bacterial septa. These results indicate that the lytR gene is involved in cell wall synthesis in S. pyognes.

Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope.

Bacteria of the order Corynebacteriales including pathogens such as <i>Mycobacterium tuberculosis</i> and <i>Corynebacterium diphtheriae</i> are characterized by their complex, multi-layered envelope. In addition to a peptidoglycan layer, these organisms possess an additional polysaccharide layer made of arabinogalactan and an outer membrane layer composed predominantly of long-chain fatty acids called mycolic acids. This so-called mycolata envelope structure is both a potent barrier against antibiotic entry into cells and a target of several antibacterial therapeutics. A better understanding of the mechanisms underlying mycolata envelope assembly therefore promises to reveal new ways of disrupting this unique structure for the development of antibiotics and antibiotic potentiators. Because they engage with receptors on the cell surface during infection, bacteriophages have long been used as tools to uncover important aspects of host envelope assembly. However, surprisingly little is known about the interactions between Corynebacteriales phages and their hosts. We therefore made use of the phages Cog and CL31 that infect <i>Corynebacterium glutamicum</i> (<i>Cglu</i>), a model member of the Corynebacteriales, to discover host factors important for phage infection. A high-density transposon library of <i>Cglu</i> was challenged with these phages followed by transposon sequencing to identify resistance loci. The analysis identified an important role for mycomembrane proteins in phage infection as well as components of the arabinogalactan and mycolic acid synthesis pathways. Importantly, the approach also implicated a new gene (<i>cgp_0396</i>) in the process of arabinogalactan modification and identified a conserved new factor (AhfA, Cpg_0475) required for mycolic acid synthesis in <i>Cglu</i>.

The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis.

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

Atomic view of the HIV-1 matrix lattice; implications on virus assembly and envelope incorporation

During the late phase of HIV type 1 (HIV-1) infection cycle, the virally encoded Gag polyproteins are targeted to the inner leaflet of the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag binding to the PM is mediated by interactions of the N-terminally myristoylated matrix (myrMA) domain with phosphatidylinositol 4,5-bisphosphate. Formation of a myrMA lattice on the PM is an obligatory step for the assembly of immature HIV-1 particles and envelope (Env) incorporation. Atomic details of the myrMA lattice and how it mediates Env incorporation are lacking. Herein, we present the X-ray structure of myrMA at 2.15 Å. The myrMA lattice is arranged as a hexamer of trimers with a central hole, thought to accommodate the C-terminal tail of Env to promote incorporation into virions. The trimer–trimer interactions in the lattice are mediated by the N-terminal loop of one myrMA molecule and α-helices I–II, as well as the 310 helix of a myrMA molecule from an adjacent trimer. We provide evidence that substitution of MA residues Leu13 and Leu31, previously shown to have adverse effects on Env incorporation, induced a conformational change in myrMA, which may destabilize the trimer–trimer interactions within the lattice. We also show that PI(4,5)P2 is capable of binding to alternating sites on MA, consistent with an MA–membrane binding mechanism during assembly of the immature particle and upon maturation. Altogether, these findings advance our understanding of a key mechanism in HIV-1 particle assembly.

Genome-Directed Cell Nucleus Assembly.

Simple SummarySpeckles and other nuclear bodies, the nucleolus and perinucleolar zone, transcription/replication factories and the lamina-associated compartment, serve as a structural basis for various genomic functions. In turn, genome activity and specific chromatin 3D organization directly impact the integrity of intranuclear assemblies, initiating/facilitating their formation and dictating their composition. Thus, the large-scale nucleus structure and genome activity mutually influence each other. The cell nucleus is frequently considered a compartment in which the genome is placed to protect it from external forces. Here, we discuss the evidence demonstrating that the cell nucleus should be considered, rather, as structure built around the folded genome. Decondensing chromosomes provide a scaffold for the assembly of the nuclear envelope after mitosis, whereas genome activity directs the assembly of various nuclear compartments, including nucleolus, speckles and transcription factories.The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA’s genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the “box” in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses.

The Cell Biology of Heterochromatin.

A conserved feature of virtually all higher eukaryotes is that the centromeres are embedded in heterochromatin. Here we provide evidence that this tight association between pericentric heterochromatin and the centromere is essential for proper metaphase exit and progression into telophase. Analysis of chromosome rearrangements that separate pericentric heterochromatin and centromeres indicates that they must remain associated in order to balance Cohesin/DNA catenation-based binding forces and centromere-based pulling forces during the metaphase–anaphase transition. In addition, a centromere embedded in heterochromatin facilitates nuclear envelope assembly around the entire complement of segregating chromosomes. Because the nuclear envelope initially forms on pericentric heterochromatin, nuclear envelope formation proceeds from the pole, thus providing time for incorporation of lagging and trailing chromosome arms into the newly formed nucleus. Additional analysis of noncanonical mitoses provides further insights into the functional significance of the tight association between heterochromatin and centromeres.

The LCP Family Protein, Psr, Is Required for Cell Wall Integrity and Virulence in Streptococcus agalactiae.

A robust cell envelope is the first line of protection for an infecting pathogen when encountering the immune defense of its host. In Gram-positive organisms, LytR-CpsA-Psr (LCP) family proteins play a major role in the synthesis and assembly of the cell envelope. While these proteins could be considered for potential new drug targets, not enough is known about how they function to support the integrity of the cell wall. Streptococcus agalactiae (group B streptococcus or GBS) is known to encode at least three LCP family proteins, including CpsA, LytR (BrpA) and Psr. Using strains of GBS that have mutations in two of the three LCP proteins, we were able to determine a role for these proteins in GBS cell wall integrity. The results presented here demonstrate that the absence of Psr results in a decreased growth rate, decreased viability over time, inconsistent cocci morphology and diminished cell wall integrity, as well as an increased penicillin susceptibility, decreased capsule levels and attenuation in virulence in a zebrafish model of infectious disease. A strain that is missing two of the LCP family proteins, CpsA and Psr, exhibits an increase in these defective phenotypes, indicating that CpsA and Psr are partially redundant in function.

An ESCRT/VPS4 Envelopment Trap To Examine the Mechanism of Alphaherpesvirus Assembly and Transport in Neurons.

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)-tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFP-VPS4-EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. IMPORTANCE Alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), are pathogens of the nervous system. They replicate in the nerve cell body and then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells; however, key aspects of how these viruses travel along axons remain controversial. Here, we test two alternative mechanisms for transport, the married and separate models, by blocking envelope assembly, a critical step in viral egress. When we arrest formation of the viral envelope using a mutated component of the cellular ESCRT apparatus, we find that entry of viral particles into axons is blocked in some types of neurons but not others. This approach allows us to determine whether envelope assembly occurs prior to entry of viruses into axons or afterwards and, thus, to distinguish between the alternative models for viral transport.

Chromosome length and gene density contribute to micronuclear membrane stability.

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

Mitotic waves in an import-diffusion model with multiple nuclei in a shared cytoplasm

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.

Inherited nuclear pore substructures template post-mitotic pore assembly.

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.

Mus musculus Barrier-To-Autointegration Factor 2 (Banf2) is Not Essential for Spermatogenesis or Fertility

The barrier-to-autointegration factor (BAF) is widely expressed in most human tissues and plays a critical role in chromatin organization, nuclear envelope assembly, gonadal development, and embryonic stem cell self-renewal. Complete loss of BAF has been shown to lead to embryonic lethality and gonadal defects. The BAF paralog, namely, barrier-to-autointegration factor 2 (BANF2), exhibits a testis-predominant expression pattern in both humans and mice. Unlike BAF, it may cause isolated male infertility. Therefore, we used the CRISPR/Cas9 system to generate Banf2-knockout mice to further study its function in spermatogenesis. Unexpectedly, knockout mice did not show any detectable abnormalities in histological structure of the testis, epididymis, ovary, and other tissues, and exhibited normal fertility, indicating that Banf2 is not essential for mouse spermatogenesis and fertility.

Genome-wide identification of Ran GTPase family genes from wheat (T. aestivum) and their expression profile during developmental stages and abiotic stress conditions

Maintenance of growth is important for sustaining yield under stress conditions. Hence, identification of genes involved in cell division and growth under abiotic stress is utmost important. Ras-related nuclear protein (Ran) is a small GTPase required for nucleocytoplasmic transport, mitotic progression, and nuclear envelope assembly in plants. In the present study, two Ran GTPase genes TaRAN1 and TaRAN2 were identified though genome-wide analysis in wheat (T. aestivum). Comparative analysis of Ran GTPases from wheat, barley, rice, maize, sorghum, and Arabidopsis revealed similar gene structure within phylogenetic clades and highly conserved protein structure. Expression analysis from expVIP platform showed ubiquitous expression of TaRAN genes across tissues and developmental stages. Under biotic and abiotic stresses, TaRAN1 expression was largely unaltered, while TaRAN2 showed stress specific response. In qRT-PCR analysis, TaRAN1 showed significantly higher expression as compared to TaRAN2 in shoot and root at seedling, vegetative, and reproductive stages. During progressive drought stress, TaRAN1 and TaRAN2 expression increase during early stress and restored to control level expression at higher stress levels in shoot. The steady-state level of transcripts was maintained to that of control in roots under drought stress. Under cold stress, expression of both the TaRAN genes decreased significantly at 3 h and became similar to control at 6 h in shoots, while salt stress significantly reduced the expression of TaRAN genes in shoots. The analysis suggests differential regulation of TaRAN genes under developmental stages and abiotic stresses. Delineating the molecular functions of Ran GTPases will help unravel the mechanism of stress induced growth inhibition in wheat.