Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae. Here Mitograph was assessed on five different mammalian cells types, all of which were accurately segmented by MitoGraph analysis. MitoGraph also successfully differentiated between distinct mitochondrial morphologies that ranged from entirely fragmented to hyper-elongated. General recommendations are also provided for confocal imaging of labeled mitochondria (using mito-YFP, MitoTracker dyes and immunostaining parameters). Widespread adoption of MitoGraph will help achieve a long-sought goal of consistent and reproducible quantification of mitochondrial morphology.

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The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by NMR that Par-6 binds a Crumbs carboxyl terminal peptide and report the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.

An academic chemical screening approach was developed by using 2D protein-detected NMR, and a 352-chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric-difference intensity analysis (DIA)-was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3-1.6 mm. DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.