Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind cooperatively to actin and regulate myosin.

Tropomyosin (Tm) is an evolutionarily conserved ?-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilising actin-filaments and regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single essential Tm, Cdc8, which can be acetylated on its amino terminal methionine to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with amino terminal substitutions to explore how stability of the Cdc8-polymer overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown the stability of the amino terminal of the Cdc8 ?-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased amino-terminal stability, dimers of which are capable of forming Tm-polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild type, and was unable to regulate actomyosin interactions. The data presented here are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin-filament, consistent with a cooperative model for Tm binding to actin

Formins Determine the Functional Properties of Actin Filaments in Yeast

The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and func- tional properties of the actin polymer. However, the mecha- nism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filament’s ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create

Characterisation and functional analysis of fission yeast tropomyosin mutants and development of quantum dot-antibody conjugates for cellular imaging

Tropomyosin (Tm) is an evolutionarily conserved dimeric a-helical coiled coil protein, which interacts end-to-end to form polymers capable of associating with and stabilising actin-filaments and thereby regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single Tm, Cdc8, an essential protein which can be acetylated on its amino terminus to increase its affinity for actin and enhance its ability to regulate myosin function. During this study extensive analyses on the physical properties of acetylated and unacetylated Cdc8 protein, together with a series of novel amino terminal Cdc8 mutants were undertaken in an attempt to explore the effects of amino terminal modification on the Cdc8 protein. In addition, a series of experiments were undertaken to develop fluorescent quantum dot (QD)-antibody (IgG) conjugates for visualising Cdc8 localisation in S. pombe cells. Modifications to the amino terminus altered the stability of the Cdc8 protein, its ability to form end-to-end interactions and its affinity for actin. Changes in actin affinity were reflected in the ability of the Cdc8 proteins to regulate myosin S1 ATPase activity. Despite changes to their biochemical properties, Cdc8 proteins expressed in a temperature sensitive S. pombe strain were capable of complementing function at the restrictive temperature. However, when expressed in a Naacetyltransferase deficient S. pombe strain, the mutant Cdc8 proteins were unable to rescue the growth defects associated with this strain. The QD-IgG conjugates produced during this study produced superior images when compared to organic fluorophores and were significantly more resistant to photobleaching. This work has provided insights into the importance of acetylation and the structure of the amino terminus for the function of fission yeast Tm and has highlighted the importance of reaction stoichiometry, the difficulties arising from non-specific binding and quenching of fluorescence intensity when coupling QDs to IgG complexes

Dark photon superradiance: Electrodynamics and multimessenger signals

We study the electrodynamics of a kinetically mixed dark photon cloud that forms through superradiance around a spinning black hole, and design strategies to search for the resulting multimessenger signals. A dark photon superradiance cloud sources a rotating dark electromagnetic field which, through kinetic mixing, induces a rotating visible electromagnetic field. Standard model charged particles entering this field initiate a transient phase of particle production that populates a plasma inside the cloud and leads to a system which shares qualitative features with a pulsar magnetosphere. We study the electrodynamics of the dark photon cloud with resistive magnetohydrodynamics methods applicable to highly magnetized plasma, adapting techniques from simulations of pulsar magnetospheres. We identify turbulent magnetic field reconnection as the main source of dissipation and electromagnetic emission, and compute the peak luminosity from clouds around solar-mass black holes to be as large as $10^{43}$ erg/s for open dark photon parameter space. The emission is expected to have a significant X-ray component and is potentially periodic, with period set by the dark photon mass. The luminosity is comparable to the brightest X-ray sources in the Universe, allowing for searches at distances of up to hundreds of Mpc with existing telescopes. We discuss observational strategies, including targeted electromagnetic follow-ups of solar-mass black hole mergers and targeted continuous gravitational wave searches of anomalous pulsars.Comment: 56 pages, 27 figures, updated to the journal versio

Mitophagy and the therapeutic clearance of damaged mitochondria for neuroprotection

Mitochondria are the foremost producers of the cellular energy currency ATP. They are also a significant source of reactive oxygen species and an important buffer of intracellular calcium. Mitochondrial retrograde signals regulate energy homeostasis and pro-survival elements whereas anterograde stimuli can trigger programmed cell death. Maintenance of a healthy, functional mitochondria network is therefore essential, and several mechanisms of mitochondrial quality control have been described. Mitochondrial dysfunction is linked to several neurodegenerative conditions including Parkinson, and Huntingdon diseases as well as Amyotrophic lateral sclerosis. Understanding the mechanisms governing mitochondrial quality control may reveal novel strategies for pharmacological intervention and disease therapy

The Threshold of Rural Placement Frequency and Duration: A Repeated Cross-Sectional Study Examining Rural Career Aspirations Among Student Nurses

Aim: This study aimed to explore what changes rural placement had on the perceptions of nursing students and the impact of placement frequency and duration on student considerations for rural practice. Background: A strong rural healthcare workforce is a global concern and has led countries to look for creative ways to address this challenge. One approach is to train more health professionals, however, nursing students who grew up or lived in metropolitan or urbanised areas are suggested to be less inclined to pursue a rural career. As such it is posited that recurrent exposure to rural settings may exert a positive impact on future intention for rural practice. However, there is a need to explore the specific thresholds related to both the frequency and duration of rural placement exposure, as well as the cumulative impact multiple rural placements may have on the intention to engage in rural practice. Design: A repeated cross-sectional design. Methods: All nursing students from an Australian regional university were invited to complete an online questionnaire between 2019 and 2023. Demographic and placement specific questions were included. A modified version of the Nursing Community Apgar tool also measured the importance of key variables in rural career decision-making. Data were analysed using independent sample t-tests and one-way ANOVAs. Significance was determined at two-tailed p≤.05. Results: Among the 835 respondents (response rate 15.4%), the average number and duration of rural placements was 2.45 placements and 3.01 weeks respectively. Rural placements did not have an impact on students who resided rurally or regionally. However, among metropolitan students who had experienced more than three rural placements, or more than sixteen cumulative weeks of placement, were significantly more likely to consider rural employment. Greater number of rural placements and longer cumulative duration had the greatest impact. Conclusion: Issues related to the nursing rural workforce are dynamic and complex. Understanding the unique drivers that improve the rural experiences among students, particularly metropolitan students, can have an impact on decision-making to pursue employment in rural environments. Importantly, whilst professional and clinical motivation and experiences are influential factors, the socialisation, environment and community features are essential elements that influence students’ decisions to pursue a career in rural practice. Undertaking a nuanced approach that facilitates rural practice understanding among students may help shape future employment decision-making

Blueberry firmness: a review of the textural and mechanical properties used in quality evaluations

Firmness is an important parameter for fresh blueberries as it influences the quality perceived by consumers and postharvest storage potential. However, the blueberry research community has not yet identified a universal standard method that can evaluate firmness for quality purposes. Different mechanical tests have been considered, offering different perspectives on this quality trait. This review summarises the most common methods previously used to evaluate textural and mechanical properties of fresh blueberries as influenced by pre- and postharvest factors. In addition, this review intends to assist the blueberry research community and commercial supply chain when selecting suitable methods to measure blueberry firmness as a fruit quality response. Different research initiatives to develop, optimize or standardise instrumental methods to assess blueberry firmness and relate to consumer sensory perception are reviewed. Mechanical parameters obtained by compression tests are the most previously used techniques to evaluate the influence of genotype, maturity, calcium, and postharvest management on blueberry firmness or to relate to sensory descriptors. However, standardising operational settings (e.g., compression distance, loading speed, and calculation procedures) is required to make results comparable across data collection conditions. Whether other mechanical test methods such as penetration or a combination of tests can better characterise blueberry quality or the relationship with consumer acceptance remains unknown and is worth studyin

Adult stem cell activity in naked mole rats for long-term tissue maintenance

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in their native in vivo state, we find an expanded pool of Lgr5+ cells in NMRs, and these cells specifically at the crypt base (Lgr5+CBC) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5+CBC cells. Instead of entering quiescence (G0), NMR Lgr5+CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1