Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast.

Puf3p binds preferentially to messenger RNAs (mRNAs) for nuclear-encoded mitochondrial proteins. We find that Puf3p localizes to the cytosolic face of the mitochondrial outer membrane. Overexpression of PUF3 results in reduced mitochondrial respiratory activity and reduced levels of Pet123p, a protein encoded by a Puf3p-binding mRNA. Puf3p levels are reduced during the diauxic shift and growth on a nonfermentable carbon source, conditions that stimulate mitochondrial biogenesis. These findings support a role for Puf3p in mitochondrial biogenesis through effects on mRNA interactions. In addition, Puf3p links the mitochore, a complex required for mitochondrial-cytoskeletal interactions, to the Arp2/3 complex, the force generator for actin-dependent, bud-directed mitochondrial movement. Puf3p, the mitochore, and the Arp2/3 complex coimmunoprecipitate and have two-hybrid interactions. Moreover, deletion of PUF3 results in reduced interaction between the mitochore and the Arp2/3 complex and defects in mitochondrial morphology and motility similar to those observed in Arp2/3 complex mutants. Thus, Puf3p is a mitochondrial protein that contributes to the biogenesis and motility of the organelle.

Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae.

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64-labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as "conveyor belts" for retrograde movement and delivery of actin patches/endosomes to FM4-64-labeled internal compartments.

Factors and Behaviors Related to the Promotion of Pediatric Hospital Medicine Fellow Autonomy: A Qualitative Study of Faculty.

OBJECTIVE: To identify factors that influence faculty to promote or reduce the expression of autonomy for pediatric hospital medicine (PHM) fellows and describe behaviors faculty employ to support fellow autonomy in the clinical setting. METHODS: This was a multicenter, qualitative study using semistructured interviews with core faculty in PHM fellowships. Data were transcribed verbatim and analyzed using a phenomenological approach. Each transcript was coded independently by 2 trained reviewers who then met to reconcile differences. Codes were identified using an iterative approach and organized into themes. Investigators engaged in peer debriefing during data collection, and member checking confirmed the results. RESULTS: Interviews were conducted December 2016 to January 2017 with 20 faculty from 5 PHM fellowships. Most participants were female (12, 60%) and assistant (13, 65%) or associate (6, 30%) professors. Data analysis yielded 6 themes. Themes reflect the importance of faculty experience, style, and approach to balancing patient care with education in the provision of autonomy for PHM fellows. Faculty appreciation for the role of autonomy in medical education, investment in their roles as educators, and investment in PHM fellowship training are also influential factors. Finally, fellow clinical, educational, leadership, and communication skills influence the provision of autonomy. Faculty employ various levels of supervision, scaffolding techniques, and direct observation with feedback to support fellow autonomy. Professional development was considered essential for developing these skills. CONCLUSIONS: We identified 6 themes related to faculty provision of autonomy to PHM fellows, as well as strategies employed by faculty to support fellow autonomy.

Live cell imaging of mitochondrial movement along actin cables in budding yeast.

BACKGROUND: Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

Mitochondrial inheritance is required for MEN-regulated cytokinesis in budding yeast.

Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p, and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Delta gem1Delta) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of beta-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile-ring closure, but does not inhibit contractile-ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation, or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Delta cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Delta and mmr1Delta gem1Delta cells and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Delta cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.