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1. Alpha‐arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

   https://doi.org/10.1111/boc.202100007  

   Robinson, Benjamin P. ; Hawbaker, Sarah ; Chiang, Annette ; Jordahl, Eric M. ; Anaokar, Sanket ; Nikiforov, Alexiy ; Bowman, II, Ray W. ; Ziegler, Philip ; McAtee, Ceara K. ; Patton‐Vogt, Jana ; et al ( October 2021 , Biology of the Cell)

   Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B‐type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools.GIT1gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasingGIT1expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization.

   We find that the α‐arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α‐arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI‐treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs inaly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α‐arrestin Aly1 by the protein phosphatase calcineurin improves steady‐state and substrate‐induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α‐arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol‐3‐phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [³H]‐inositol‐labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a‐arrestins in this regulation.

   The α‐arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance.

   To our knowledge, this is the first example of α‐arrestin regulation of phosphatidyliniositol‐3‐phosphate levels. In future studies it will be exciting to determine if other α‐arrestins similarly alter PI and PIPs to change the cellular landscape.

    

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2. Two transporters mobilize magnesium from vacuolar stores to enable plant acclimation to magnesium deficiency

   https://doi.org/10.1093/plphys/kiac330  

   Tang, Ren-Jie ; Yang, Yang ; Yan, Yu-Wei ; Mao, Dan-Dan ; Yuan, Hong-Mei ; Wang, Chao ; Zhao, Fu-Geng ; Luan, Sheng ( July 2022 , Plant Physiology)

   Magnesium (Mg) is an essential metal for chlorophyll biosynthesis and other metabolic processes in plant cells. Mg is largely stored in the vacuole of various cell types and remobilized to meet cytoplasmic demand. However, the transport proteins responsible for mobilizing vacuolar Mg2+ remain unknown. Here, we identified two Arabidopsis (Arabidopsis thaliana) Mg2+ transporters (MAGNESIUM TRANSPORTER 1 and 2; MGT1 and MGT2) that facilitate Mg2+ mobilization from the vacuole, especially when external Mg supply is limited. In addition to a high degree of sequence similarity, MGT1 and MGT2 exhibited overlapping expression patterns in Arabidopsis tissues, implying functional redundancy. Indeed, the mgt1 mgt2 double mutant, but not mgt1 and mgt2 single mutants, showed exaggerated growth defects as compared to the wild type under low-Mg conditions, in accord with higher expression levels of Mg-starvation gene markers in the double mutant. However, overall Mg level was also higher in mgt1 mgt2, suggesting a defect in Mg2+ remobilization in response to Mg deficiency. Consistently, MGT1 and MGT2 localized to the tonoplast and rescued the yeast (Saccharomyces cerevisiae) mnr2Δ (manganese resistance 2) mutant strain lacking the vacuolar Mg2+ efflux transporter. In addition, disruption of MGT1 and MGT2 suppressed high-Mg sensitivity of calcineurin B-like 2 and 3 (cbl2 cbl3), a mutant defective in vacuolar Mg2+ sequestration, suggesting that vacuolar Mg2+ influx and efflux processes are antagonistic in a physiological context. We further crossed mgt1 mgt2 with mgt6, which lacks a plasma membrane MGT member involved in Mg2+ uptake, and found that the triple mutant was more sensitive to low-Mg conditions than either mgt1 mgt2 or mgt6. Hence, Mg2+ uptake (via MGT6) and vacuolar remobilization (through MGT1 and MGT2) work synergistically to achieve Mg2+ homeostasis in plants, especially under low-Mg supply in the environment.

    

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3. Elucidating the regulatory mechanism of Swi1 prion in global transcription and stress responses

   https://doi.org/10.1038/s41598-020-77993-0  

   Du, Zhiqiang ; Regan, Jeniece ; Bartom, Elizabeth ; Wu, Wei-Sheng ; Zhang, Li ; Goncharoff, Dustin Kenneth ; Li, Liming ( December 2020 , Scientific Reports)

   Transcriptional regulators are prevalent among identified prions inSaccharomyces cerevisiae, however, it is unclear how prions affect genome-wide transcription.We show here that the prion ([SWI⁺]) and mutant (swi1∆)forms of Swi1, a subunit of the SWI/SNF chromatin-remodeling complex, confer dramatically distinct transcriptomic profiles. In [SWI⁺] cells, genes encoding for 34 transcription factors (TFs) and 24 Swi1-interacting proteins can undergo transcriptional modifications. Several TFs show enhanced aggregation in [SWI⁺] cells. Further analyses suggest that such alterations are key factors in specifying the transcriptomic signatures of [SWI⁺] cells. Interestingly,swi1∆and [SWI⁺] impose distinct and oftentimes opposite effects on cellular functions. Translation-associated activities, in particular, are significantly reduced inswi1∆cells. Although bothswi1∆and [SWI⁺] cells are similarly sensitive to thermal, osmotic and drought stresses, harmful, neutral or beneficial effects were observed for a panel of tested chemical stressors. Further analyses suggest that the environmental stress response (ESR) is mechanistically different betweenswi1∆and [SWI⁺] cells—stress-inducible ESR (iESR) are repressed by [SWI⁺] but unchanged byswi1∆while stress-repressible ESR (rESR) are induced by [SWI⁺] but repressed byswi1∆. Our work thus demonstrates primarily gain-of-function outcomes through transcriptomic modifications by [SWI⁺] and highlights a prion-mediated regulation of transcription and phenotypes in yeast.

    

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4. Time-resolved microfluidics unravels individual cellular fates during double-strand break repair

   https://doi.org/10.1186/s12915-022-01456-3  

   Vertti-Quintero, Nadia ; Levien, Ethan ; Poggi, Lucie ; Amir, Ariel ; Richard, Guy-Franck ; Baroud, Charles N. ( December 2022 , BMC Biology)

   Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such asSaccharomyces cerevisiaehave been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair.

   We used engineeredS. cerevisiaecells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80–150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair.

   Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.

    

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5. Cell behaviors underlying Myxococcus xanthus aggregate dispersal

   https://doi.org/10.1128/msystems.00425-23  

   Murphy, Patrick ; Comstock, Jessica ; Khan, Trosporsha ; Zhang, Jiangguo ; Welch, Roy ; Igoshin, Oleg A. ( September 2023 , mSystems)

   Rodríguez-Verdugo, Alejandra (Ed.)

   The soil bacteriumMyxococcus xanthusis a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a secondM. xanthusstrain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle ofM. xanthus.

   Understanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacteriumMyxococcus xanthusforms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

    

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