Structure of the MIND Complex Defines a Regulatory Focus for Yeast Kinetochore Assembly.

Kinetochores connect centromeric nucleosomes with mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND complex (Mtw1, Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.

Probing the spatial organization of bacteriochlorophyll C by solid-state nuclear magnetic resonance.

Green sulfur bacteria, which live in extremely low-light environments, use chlorosomes to harvest light. A chlorosome is the most efficient, and arguably the simplest, light-harvesting antenna complex, which contains hundreds of thousands of densely packed bacteriochlorophylls (BChls). To harvest light efficiently, BChls in a chlorosome form supramolecular aggregates; thus, it is of great interest to determine the organization of the BChls in a chlorosome. In this study, we conducted a (13)C solid-state nuclear magnetic resonance and Mg K-edge X-ray absorption analysis of chlorosomes from wild-type Chlorobaculum tepidum. The X-ray absorption results indicated that the coordination number of the Mg in the chlorosome must be >4, providing evidence that electrostatic interactions formed between the Mg of a BChl and the carbonyl group or the hydroxyl group of the neighboring BChl molecule. According to the intermolecular distance constraints obtained on the basis of (13)C homonuclear dipolar correlation spectroscopy, we determined that the molecular assembly of BChls is dimer-based and that the hydrogen bonds among the BChls are less extensive than commonly presumed because of the twist in the orientation of the BChl dimers. This paper also reports the first (13)C homonuclear correlation spectrum acquired for carotenoids and lipids-which are minor, but crucial, components of chlorosomes-extracted from wild-type Cba. tepidum.

Challenges and priorities for suicide prevention in Southeast Asia: Insights from the Partnerships for Life regional workshop on suicide prevention.

Compared to the West, suicide prevention in the Southeast Asian (SEA) region is challenging due to resource constraints, a relatively greater contribution of social compared to psychological factors, and low levels of general awareness coupled with high stigma around suicide and mental illness. Collaboration and knowledge sharing are essential to circumvent these challenges. The Partnerships for Life (PfL) initiative of the International Association of Suicide Prevention aims to enhance knowledge sharing, foster collaboration between nations, and support the development and implementation of evidence-informed approaches to suicide prevention. In February 2024, the SEA region of the PfL conducted the first regional workshop on suicide prevention, in which representatives from 10 out of 12 SEA nations participated. In this paper, we outline the key priorities, challenges, strengths, and opportunities for suicide prevention in the region with a view to inform resource-effective suicide prevention strategies that have optimal utility and uptake.

Temperature shift effect on the Chlorobaculum tepidum chlorosomes.

Chlorobaculum [Cba.] tepidum is known to grow optimally at 48-52 °C and can also be cultured at ambient temperatures. In this paper, we prepared constant temperature, temperature shift, and temperature shift followed by backshift cultures and investigated the intrinsic properties and spectral features of chlorosomes from those cultures using various approaches, including temperature-dependent measurements on circular dichroism (CD), UV-visible, and dynamic light scattering. Our studies indicate that (1) chlorosomes from constant temperature cultures at 50 and 30 °C exhibited more resistance to heat relative to temperature shift cultures; (2) as temperature increases bacteriochlorophyll c (BChl c) in chlorosomes is prone to demetalation, which forms bacteriopheophytin c, and degradation under aerobic conditions. Some BChl c aggregates inside reduced chlorosomes prepared in low-oxygen environments can reform after heat treatments; (3) temperature shift cultures synthesize and incorporate more BChl c homologs with a smaller substituent at C-8 on the chlorin ring and less BChl c homologs with a larger long-chain alcohol at C-17(3) versus constant-temperature cultures. We hypothesize that the long-chain alcohol at C-17(3) (and perhaps together with the substituent at C-8) may account for thermal stability of chlorosomes and the substituent at C-8 may assist self-assembling BChls; and (4) while almost identical absorption spectra are detected, chlorosomes from different growth conditions exhibited differences in the rotational length of the CD signal, and aerobic and reduced chlorosomes also display different Qy CD intensities. Further, chlorosomes exhibited changes of CD features in response to temperature increases. Additionally, we compare temperature-dependent studies for the Cba. tepidum chlorosomes and previous studies for the Chloroflexus aurantiacus chlorosomes. Together, our work provides useful and novel insights on the properties and organization of chlorosomes.

Cryoelectron Microscopy Structure of a Yeast Centromeric Nucleosome at 2.7 Å Resolution.

Kinetochores mediate chromosome segregation during cell division. They assemble on centromeric nucleosomes and capture spindle microtubules. In budding yeast, a kinetochore links a single nucleosome, containing the histone variant Cse4CENP-A instead of H3, with a single microtubule. Conservation of most kinetochore components from yeast to metazoans suggests that the yeast kinetochore represents a module of the more complex metazoan arrangements. We describe here a streamlined protocol for reconstituting a yeast centromeric nucleosome and a systematic exploration of cryo-grid preparation. These developments allowed us to obtain a high-resolution cryoelectron microscopy reconstruction. As suggested by previous work, fewer base pairs are in tight association with the histone octamer than there are in canonical nucleosomes. Weak binding of the end DNA sequences may contribute to specific recognition by other inner kinetochore components. The centromeric nucleosome structure and the strategies we describe will facilitate studies of many other aspects of kinetochore assembly and chromatin biochemistry.

Glycine Cleavage Powers Photoheterotrophic Growth of Chloroflexus aurantiacus in the Absence of H 2.

Chloroflexus aurantiacus is an anoxygenic phototrophic bacterium. Its unique CO2 fixation pathway and primitive light-harvesting antenna complexes have attracted extensive research attentions. In this work, we investigated the photoheterotrophic growth of C. aurantiacus J-10-fl using acetate [at 55°C and without H2(g)]. The results indicate that glycine can promote anaerobic biomass production in a minimal medium by threefold to fivefold. Via (13)C-metabolite analysis, we observed that glycine was involved in serine synthesis. Instead of being used as a major carbon source, glycine was degraded to produce C1 units and NAD(P)H. Tracer experiments also suggest that photoheterotrophic cultures growing with a exogenous glycine source exhibited capabilities of assimilating CO2 via multiple routes (including the 3-hydroxypropionate pathway). Finally, glycylglycine, a commonly used culture buffer, also significantly enhanced photoheterotrophic growth of C. aurantiacus, probably due to its thermal or enzymatic breakdown to glycine.

Chromatic acclimation and population dynamics of green sulfur bacteria grown with spectrally tailored light.

Living organisms have to adjust to their surrounding in order to survive in stressful conditions. We study this mechanism in one of most primitive creatures - photosynthetic green sulfur bacteria. These bacteria absorb photons very efficiently using the chlorosome antenna complexes and perform photosynthesis in extreme low-light environments. How the chlorosomes in green sulfur bacteria are acclimated to the stressful light conditions, for instance, if the spectrum of light is not optimal for absorption, is unknown. Studying Chlorobaculum tepidum cultures with far-red to near-infrared light-emitting diodes, we found that these bacteria react to changes in energy flow by regulating the amount of light-absorbing pigments and the size of the chlorosomes. Surprisingly, our results indicate that the bacteria can survive in near-infrared lights capturing low-frequency photons by the intermediate units of the light-harvesting complex. The latter strategy may be used by the species recently found near hydrothermal vents in the Pacific Ocean.