Biology subjects

Anand, R.

Publications and source records attributed to Anand, R..

27 records · Page 2Linked to original sources

bioRxiv2021

Interactive effects of microplastic pollution and heat stress on reef-building corals

Plastic pollution is an emerging stressor that increases pressure on ecosystems such as coral reefs that are already challenged by climate change. However, the effect of plastic pollution in combination with global warming is largely unknown. Thus, the goal of this study was to determine the cumulative effect of microplastic pollution with that of global warming on reef-building coral species and to compare the severity of both stressors. For this, we conducted a series of three controlled laboratory experiments and exposed a broad range of coral species (Acropora muricata, Montipora digitata, Porites lutea, Pocillopora verrucosa, and Stylophora pistillata) to microplastic particles in a range of concentrations (2.5-2,500 particles L-1) and mixtures (from different industrial sectors) at ambient temperatures and in combination with heat stress. We show that microplastic can occasionally have a negative effect on the corals thermal tolerance. In comparison to heat stress, however, microplastic constitutes a minor stressor. While heat stress led to decreased photosynthetic efficiency of algal symbionts, and increased bleaching, tissue necrosis, and mortality, treatment with microplastic particles had only minor effects on the physiology and health of the tested coral species at ambient temperatures. These findings underline that while efforts to reduce plastic pollution should continue, they should not replace more urgent efforts to halt global warming, which are immediately needed to preserve remaining coral reef ecosystems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/454458v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@c2eeb0org.highwire.dtl.DTLVardef@1c49ea6org.highwire.dtl.DTLVardef@18bab59org.highwire.dtl.DTLVardef@9e952b_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2021

NECAB2 orchestrates an endosomal pathway of mitochondrial quality control at striatal synapses

Synaptic signaling depends on ATP generated by mitochondria. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction and thus requires efficient mitochondrial quality control and repair. We found that global knockout of the neuronal calcium-binding protein 2 (NECAB2) in the mouse causes loss of striatal synapses and behavioral phenotypes related to striatal dysfunction such as reduced motivation and altered sensory gating. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller. They are characterized by increased respiration and superoxide production resulting in oxidative stress. This accumulation of dysfunctional mitochondria is caused by a defective assembly of mitochondria with early endosomes in a pathway that involves the small GTPase Rab5 and its guanine nucleotide exchange factor Alsin/ALS2. NECAB2 therefore participates in an endosomal pathway of mitochondrial stress response and repair important for striatal function.

neuroscience↗

bioRxiv2020

Protease OMA1 Modulates Mitochondrial Bioenergetics and Ultrastructure through Dynamic Association with MICOS Complex

Remodeling of mitochondrial ultrastructure is a complex dynamic process that is critical for a variety of mitochondrial functions and apoptosis. Although the key regulators of this process - mitochondrial contact site and cristae junction organizing system (MICOS) and GTPase Optic Atrophy 1 (OPA1) have been characterized, the mechanisms behind this regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for maintenance of contacts between the inner and outer membranes through a dynamic association with MICOS. This association is independent of OPA1, appears to be mediated via the MICOS subunit MIC60, and is important for stability of MICOS machinery and the inner-outer mitochondrial membrane contacts. We find that OMA1-MICOS relay is required for stability of respiratory supercomplexes, optimal bioenergetic output in response to cellular insults, and apoptosis. Loss of OMA1 affects these activities; remarkably it can be partially compensated for by an artificial MICOS-emulating tether protein that bridges the inner and outer mitochondrial membranes. Our data show that OMA1-mediated support of mitochondrial ultrastructure is required for maintenance of mitochondrial architecture and bioenergetics under both basal and homeostasis-challenging conditions, and suggest a previously unrecognized role for this protease in mitochondrial physiology.

biochemistry↗

bioRxiv2020

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

Homologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a chaperone to promote 3 to 5 filament growth via highly dynamic engagement with 5 filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

biophysics↗

bioRxiv2020

Regulation of the MLH1-MLH3 endonuclease in meiosis

During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny1. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the putative MLH1-MLH3 (MutL{gamma}) nuclease in generating crossovers, but mechanisms that lead to its activation were unclear2-4. Here, we have biochemically reconstituted key elements of this pro-crossover pathway. First, we show that human MSH4-MSH5 (MutS{gamma}), which was known to support crossing over5-7, binds branched recombination intermediates and physically associates with MutL{gamma}. This helps stabilize the ensemble at joint molecule structures and adjacent dsDNA. Second, we show that MutS{gamma} directly stimulates DNA cleavage by the MutL{gamma} endonuclease, which demonstrates a novel and unexpected function for MutS{gamma} in triggering crossing-over. Third, we find that MutL{gamma} activity is further stimulated by EXO1, but only when MutS{gamma} is present. Fourth, we also identify the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) as additional components of the nuclease ensemble, and show that S. cerevisiae strains expressing PIP box-mutated MutL{gamma} present striking defects in forming crossovers. Finally, we show that the MutL{gamma}-MutS{gamma}-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, the multilayered nuclease ensemble likely processes meiotic recombination intermediates by nicking dsDNA adjacent to junction points8. Since DNA nicking by MutL{gamma} is dependent on its co-factors, the asymmetric distribution of MutS{gamma} and RFC/PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This unique mode of MutL{gamma} nuclease activation might explain crossover-specific processing of Holliday junctions within the meiotic chromosomal context3, 9.

biochemistry↗

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗

bioRxiv2019

CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

BLM or WRN helicases function with the DNA2 helicase-nuclease to resect DNA doublestrand breaks and initiate homologous recombination. Upon DNA unwinding by BLM/WRN, RPA directs the DNA2 nuclease to degrade the 5-strand, revealing the 3 overhang needed for recombination. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single molecule biochemistry, we show that phosphorylated CtIP dramatically stimulates the ATP hydrolysis driven motor activity of DNA2. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. The domains of CtIP required to stimulate DNA2 are separable from those that regulate the MRN complex. These results establish that CtIP couples both MRE11-dependent short and DNA2-dependent long-range resection, and show how the motor activity of DNA2 promotes resection. Our data explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.\n\nHighlightsO_LIPhosphorylated CtIP stimulates the motor activity of DNA2\nC_LIO_LIThe activated DNA2 translocase facilitates degradation of RPA-coated ssDNA\nC_LIO_LICtIP promotes both MRN and DNA2 nucleases coupling short and long-range resection\nC_LIO_LIThe CtIP domains required to promote DNA2 and MRN are distinct and fully separable\nC_LI

biochemistry↗

bioRxiv2019

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IPs ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.

genetics↗

bioRxiv2019

Cristae undergo continuous cycles of fusion and fission in a MICOS-dependent manner

The mitochondrial inner membrane can reshape under different physiological conditions. How and at which frequency this occurs in vivo and what are the molecular players involved is unknown. Here we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that crista junctions (CJs) are dynamically fusing and dividing in a reversible and balanced manner at a timescale of seconds. CJ dynamics is strongly reduced in the absence of the MICOS subunit MIC13. Staining of the cristae membrane using different protein markers or two inner mitochondrial membrane-specific dyes revealed that cristae also undergo continuous cycles of fusion and fission. These processes are dependent on MIC13 and occur at a timescale of seconds, resembling CJ dynamics. Our data further suggest that MIC60 acts as a docking platform pioneering CJ formation. Overall, by employing a variety of advanced imaging techniques including FRAP (Fluorescence-Recovery-After Photobleaching), SPT (Single-Particle-Tracking), live-cell STED and confocal Airyscan microscopy we demonstrate that cristae undergo continuous cycles of fusion and fission in a manner that is mechanistically linked to CJ formation and dynamics.

cell biology↗