Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation.

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes.

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

The Ogura sterility-inducing protein forms a large complex without interfering with the oxidative phosphorylation components in rapeseed mitochondria.

The Ogura cytoplasmic male sterility causing protein, ORF138, was found to be part of a complex with an apparent size of over 750 kDa in the inner membrane of mitochondria of sterile plants. ORF138 did not colocalize with any of the oxidative phosphorylation complexes, nor did its presence modify their apparent size or amount, compared to samples from fertile isogenic plants. We attempted to detect potential proteins or nucleic acids that could be involved in the large ORF138 complex by 2D PAGE, immunoprecipitation and nuclease treatments of native extracts. All our results suggest that the ORF138 protein is the main, if not only, component of this large complex. The capacities of complexes I, II, IV, and ATP synthase were identical in samples from sterile and fertile plants. Isolated mitochondria from sterile plants showed a higher oxygen consumption than those from fertile plants. In vivo respiration measurements suggest that the difference in O(2) consumption measured at the organelle level is compensated at the cell/tissue level, completely in leaves, but only partially in male reproductive organs.

Mutations in three distinct loci cause resistance to peptide deformylase inhibitors in Bacillus subtilis.

Bacillus subtilis mutants with resistance against peptide deformylase inhibitors were isolated. All showed a bypass of the pathway through mutations in three genes required for formylation of Met-tRNA(fMet), fmt, folD, and glyA. glyA corresponds to a yet uncharacterized locus inducing resistance. The bypass of formylation caused robust fitness reduction but was not accompanied by alterations of the transcription profile. A subtle adaptation of the enzymes of the intermediary metabolism was observed.

Discovery and refinement of a new structural class of potent peptide deformylase inhibitors.

New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells.

The preprophase band of microtubules controls the robustness of division orientation in plants.

Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

Concerted action of the MutLß heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion.

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLß complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLß preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLß is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Biochemical and functional characterization of ORF138, a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae.

In cytoplasmic male sterility (CMS), original mitochondrial genes contribute to sex determinism by provoking pollen abortion. The function of the encoded proteins remains unclear. We studied the ORF138 protein, responsible for the 'Ogura' CMS, which is both used in hybrid seed production and present in natural populations. We analyzed the biochemical and structural properties of this protein in male-sterile plants and in E. coli. We showed that this protein spontaneously forms dimers in vitro. Truncated variants of the protein, containing either the hydrophobic or the hydrophilic moiety, also spontaneously dimerize. By fractionating mitochondria, we showed that ORF138 was strongly associated with the inner mitochondrial membrane of male-sterile plants. Our results also strongly suggest that ORF138 forms oligomers in male-sterile plant mitochondria. In E. coli, ORF138 was associated with the plasma membrane, as shown by membrane fractionation, and formed oligomers. The production of this protein strongly inhibited bacterial growth, but not by inhibiting respiration. The observed toxic effects required both the hydrophilic and hydrophobic moieties of the protein.

A protein phosphatase 2A complex spatially controls plant cell division.

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

Structure-activity relationship analysis of the peptide deformylase inhibitor 5-bromo-1H-indole-3-acetohydroxamic acid.

The lead compound 5-bromoindolyl-3-acetohydroxamic acid (10) was recently identified as a potent inhibitor of bacterial peptide deformylases (PDFs). The synthesis and associated activities of new variants were investigated at position 5 to optimize the fit at the S1' subsite and at position 1 to improve both potency and antibacterial activity. A morphomimetic series, termed "reverse-indole" was synthesized. The indole derivatives remain selective in vitro inhibitors of PDF2 over PDF1. Bromide is the best group at position 5 and cannot be replaced by bulkier substituents. In this series, an N-benzyl group at position 1 in 19 e improves the potency relative to 10. In the case of PDF1, and unlike PDF2, potency is increased as the alkyl chain becomes longer and more ramified. These data support the results of NMR footprinting experiments that were performed with (15)N-labeled Ni-PDF and the corresponding 3-acetic acid derivatives. Most of the compounds have antibacterial activities toward B. subtilis, but are inefficient toward E. coli owing to active removal by the major efflux pumps. Among the reverse-indole derivatives, 23 c, which is the exact mirror image of 19 e, shows strong potency in vitro against PDF2, but little against PDF1, although this compound displays significant antibacterial activity toward an efflux-minus mutant of E. coli. All the compounds were assessed with major pathogenic bacteria, but most of them are inefficient antibacterial agents. The reverse-indole compounds 23 a and 23 c have potency against S. pneumoniae that is similar to that of actinonin.