Parkin interacts with Ambra1 to induce mitophagy

Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy

Image-based dynamic phenotyping reveals genetic determinants of filamentation-mediated beta-lactam tolerance

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. beta-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to beta-lactam tolerance. Systematic discovery of genetic factors that affect beta-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the beta-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by beta-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after beta-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated beta-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

STUDENT CENTERED MATHEMATICS INSTRUCTION: DEVELOPING A TEACHER AND STUDENT SURVEY AND UNDERSTANDING STUDENTS’ EXPERIENCES

Over the past thirty years, policies have been enacted at the local and state levels to reform mathematics instruction to be student-centered (see Cohen & Ball, 1990; Resnick, Stein, & Coon, 2008). Despite the wave of instructional reforms, several critical gaps remain in our understanding of student-centered mathematics instruction. First, the field lacks a conceptual framework that relates the underlying theories with student-centered instructional practice in mathematics. Second, there has been very little systematic and large-scale research on the implementation and effects of student-centered mathematics instruction. Third, we know very little about how students experience and respond to the implementation of student-centered instructional reform. The dissertation studies aimed to bridge these gaps with two mixed methods studies. Study 1 proposes a conceptual framework of student-centered mathematics instruction and uses a combination of literature review, feedback from experts, and data from a large sample of urban and suburban youth (n= 2,536 students) and their mathematics teachers (n = 34) to validate a student and teacher survey of student-centered mathematics instruction. Study 2 investigates seventh grade adolescents’ experiences of their mathematics teachers implementing a reform to student-centered instruction. Specifically, the study examines the emotional experience of getting stuck in their algebra coursework and how the frequency and nature of these emotions vary by student characteristics and how students’ emotional experiences influence their sense of competence in math. The studies have important implications for our understanding of student-centered mathematics instruction. In particular, the studies suggest that students’ perspective and experiences could be important to both the implementation and effects of student-centered mathematics instruction. The studies also suggest mechanisms for differential experiences and effects of student-centered instruction for minority, low-income, and female students. Study findings are discussed in terms of implications for education research, practice, and theory

Assessing the Resolution of Methyltransferase-Mediated DNA Optical Mapping

[Image: see text] Interest in the human microbiome is growing and has been, for the past decade, leading to new insights into disease etiology and general human biology. Stimulated by these advances and in a parallel trend, new DNA sequencing platforms have been developed, radically expanding the possibilities in microbiome research. While DNA sequencing plays a pivotal role in this field, there are some technological hurdles that are yet to be overcome. Targeting of the 16S rRNA gene with amplicon sequencing, for instance, is frequently used for sample composition profiling due to its short sample-to-result time and low cost, which counterbalance its low resolution (genus to species level). On the other hand, more comprehensive methods, namely, whole-genome sequencing (WGS) and shallow shotgun sequencing, are capable of yielding single-gene- and functional-level resolution at a higher cost and much higher sample processing time. It goes without saying that the existing gap between these two types of approaches still calls for the development of a fast, robust, and low-cost analytical platform. In search of the latter, we investigated the taxonomic resolution of methyltransferase-mediated DNA optical mapping and found that strain-level identification can be achieved with both global and whole-genome analyses as well as using a unique identifier (UI) database. In addition, we demonstrated that UI selection in DNA optical mapping, unlike variable region selection in 16S amplicon sequencing, is not limited to any genomic location, explaining the increase in resolution. This latter aspect was highlighted by SCCmec typing in methicillin-resistant Staphylococcus aureus (MRSA) using a simulated data set. In conclusion, we propose DNA optical mapping as a method that has the potential to be highly complementary to current sequencing platforms

Defocused imaging of UV-driven surface-bound molecular motors

Synthetic molecular motors continue to attract great interest due to their ability to transduce energy into nanomechanical motion, the potential to do work and drive systems out-of-equilibrium. Of particular interest are unidirectional rotary molecular motors driven by chemical fuel or light. Probing the mechanistic details of their operation at the single-molecule level is hampered by the diffraction limit, which prevents the collection of dynamic positional information by traditional optical methods. Here, we use defocused wide-field imaging to examine the unidirectional rotation of individual molecular rotary motors on a quartz surface in unprecedented detail. The sequential occupation of nanomechanical states during the UV and heat-induced cycle of rotation are directly imaged in real-time. The approach will undoubtedly prove important in elucidating the mechanistic details and assessing the utility of novel synthetic molecular motors in the future

Aminobacter sp MSH1 invades sand filter community biofilms while retaining 2,6-dichlorobenzamide degradation functionality under C- and N-limiting conditions

Aminobacter sp. MSH1 is of interest for bioaugmentation of biofiltration units in drinking water treatment plants (DWTPs) due to its ability to degrade the groundwater micropollutant 2,6-dichlorobenzamide (BAM). Using a continuous flow chamber biofilm model, MSH1 was previously shown to colonize surfaces and degrade BAM at trace concentrations as low as 1 mu g/L under the oligotrophic conditions found in DWTPs. In DWTP filtration units, MSH1 has to compete with the resident biofilm microbiota for space and nutrients. Using the same model, we examined how a sand filter community (SFC) affects MSH1's BAM-degrading activity and biofilm formation under C-and N-limiting conditions when fed with trace concentrations of BAM. MSH1 was inoculated simultaneously with the SFC (co-colonization mode) or after the SFC formed a biofilm (invasion mode). MSH1 successfully established in the SFC biofilm showing growth and activity. In co-colonization mode, MSH1 decreased in number in the presence of the SFC and formed isolated colonies, while specific BAM-degradation activity increased. In the invasion mode, MSH1 also decreased in numbers in the presence of the SFC but formed mixed colonies, while specific BAM degradation was unaffected. Our results show that MSH1 invades and performs successfully in an SFC biofilm under the oligotrophic conditions of DWTPs