SRCP1 Conveys Resistance to Polyglutamine Aggregation.

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.

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission.

Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in Saccharomyces cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (KD = 12-68 µM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss-of-function and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A), demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

A conserved, noncanonical insert in FIS1 mediates TBC1D15 and DRP1 recruitment for mitochondrial fission.

Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes, yet its function in metazoans is thought divergent. Structure-based sequence alignments of FIS1 revealed a conserved, but noncanonical, three-residue insert in its first tetratricopeptide repeat (TPR) suggesting a conserved function. In vertebrates, this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of the "SKY insert," three variants were tested in HCT116 cells for altered mitochondrial morphology and recruitment of fission mechanoenzyme DRP1 and mitophagic adaptor TBC1D15. Similar to ectopically expressed wildtype FIS1, substitution of the SKY insert with alanine (AAA) fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1. In contrast, deletion variants (either ∆SKY or ∆SKYD49G) elongated mitochondrial networks with reduced mitochondrial recruitment of DRP1, despite DRP1 coimmunoprecipitates being highly enriched with ΔSKY variants. Ectopic wildtype FIS1 drove co-expressed YFP-TBC1D15 entirely from the cytoplasm to mitochondria as punctate structures concomitant with enhanced mitochondrial DRP1 recruitment. YFP-TBC1D15 co-expressed with the AAA variant further enhanced mitochondrial DRP1 recruitment, indicating a gain of function. In contrast, YFP-TBC1D15 co-expressed with deletion variants impaired mitochondrial DRP1 and YFP-TBC1D15 recruitment; however, mitochondrial fragmentation was restored. These phenotypes were not due to misfolding or poor expression of FIS1 variants, although ∆SKYD49G induced conformational heterogeneity that is lost upon deletion of the regulatory Fis1 arm, indicating SKY-arm interactions. Collectively, these results support a unifying model whereby FIS1 activity is effectively governed by intramolecular interactions between its regulatory arm and a noncanonical TPR insert that is conserved across eukaryotes.

Different Mouse Models of Nemaline Myopathy Harboring Acta1 Mutations Display Differing Abnormalities Related to Mitochondrial Biology.

ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.

Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy.

Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

Mitochondria-targeted drugs stimulate mitophagy and abrogate colon cancer cell proliferation.

Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

OBJECTIVE: We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. APPROACH AND RESULTS: Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. CONCLUSIONS: Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction.

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy.

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.

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph.

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.

A Targeted Mutation Identified through pKa Measurements Indicates a Postrecruitment Role for Fis1 in Yeast Mitochondrial Fission.

The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission.

Mitochondrial Fission Protein 1 (Fis1) and Dynamin Related Protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in S. cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 ( K D = 12-68 µM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss- and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A) demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae . These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

Fragment-based screening by protein-detected NMR spectroscopy.

Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.

The variable domain from dynamin-related protein 1 promotes liquid-liquid phase separation that enhances its interaction with cardiolipin-containing membranes.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" important for regulation. For the mitochondrial fission dynamin, dynamin-related protein 1, a regulatory role for the variable domain (VD) is demonstrated by gain- and loss-of-function mutations, yet the basis for this is unclear. Here, the isolated VD is shown to be intrinsically disordered and undergo a cooperative transition in the stabilizing osmolyte trimethylamine N-oxide. However, the osmolyte-induced state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, which appears to promote phase separation. Since dynamin-related protein 1 is found assembled into discrete punctate structures on the mitochondrial surface, the inference from the present work is that these structures might arise from a condensed state involving the VD that may enable rapid tuning of mechanoenzyme assembly necessary for fission.

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

The PARL family of mitochondrial rhomboid proteases.

Rhomboids are an ancient and conserved family of intramembrane-cleaving proteases, a small group of proteolytic enzymes capable of hydrolyzing a peptide bond within a transmembrane helix that anchors a substrate protein to the membrane. Mitochondrial rhomboids evolved in eukaryotes to coordinate a critical aspect of cell biology, the regulation of mitochondrial membranes dynamics. This function appears to have required the emergence of a structural feature that is unique among all other rhomboids: an additional transmembrane helix (TMH) positioned at the N-terminus of six TMHs that form the core proteolytic domain of all prokaryotic and eukaryotic rhomboids. This "1+6" structure, which is shared only among mitochondrial rhomboids, defines a subfamily of rhomboids with the prototypical family member being mammalian Parl. Here, we present the findings that in 11 years have elevated mitochondrial rhomboids as the gatekeepers of mitochondrial dynamics and apoptosis; further, we discuss the aspects of their biology that are bound to introduce new paradigm shifts in our understanding of how the organelle uses this unique type of protease to govern stress, signaling to the nucleus, and other key mitochondrial activities in health and disease.

Histone H3 and H4 tails play an important role in nucleosome phase separation.

Chromatin organization and its dynamic regulation are crucial in governing the temporal and spatial accessibility of DNA for proper gene expression. Disordered chains of nucleosomes comprise the basis of eukaryotic chromatin, forming higher-level organization across a range of length scales. Models of chromatin organization involving phase separation driven by chromatin-associating proteins have been proposed. More recently, evidence has emerged that nucleosome arrays can phase separate in the absence of other protein factors, yet questions remain regarding the molecular basis of chromatin phase separation that governs this dynamic nuclear organization. Here, we break chromatin down into its most basic subunit, the nucleosome core particle, and investigate phase separation using turbidity assays in conjunction with differential interference contrast microscopy. We show that, at physiologically-relevant concentrations, this fundamental subunit of chromatin undergoes phase separation. Individually removing the H3 and H4 tails abrogates phase separation under the same conditions. Taking a reductionist approach to investigate H3 and H4 tail peptide interactions in-trans with DNA and nucleosome core particles supports the direct involvement of these tails in chromatin phase separation. These results provide insight into fundamental mechanisms underlying phase separation of chromatin, which starts at the level of the nucleosome core particle, and support that long-range inter-nucleosomal interactions are sufficient to drive phase separation at nuclear concentrations. Additionally, our data have implications for understanding crosstalk between histone tails and provide a lens through which to interpret the effect of histone post-translational modifications and sequence variants. STATEMENT OF SIGNIFICANCE: Emerging models propose that chromatin organization is based in phase separation, however, mechanisms that drive this dynamic nuclear organization are only beginning to be understood. Previous focus has been on phase separation driven by chromatin-associating proteins, but this has recently shifted to recognize a direct role of chromatin in phase separation. Here, we take a fundamental approach in understanding chromatin phase separation and present new findings that the basic subunit of chromatin, the nucleosome core particle, undergoes phase separation under physiological concentrations of nucleosome and monovalent salt. Furthermore, the histone H3 and H4 tails are involved in phase separation in a manner independent of histone-associating proteins. These data suggest that H3 and H4 tail epigenetic factors may modulate chromatin phase separation.

Novel <i>DNM1L</i> variants impair mitochondrial dynamics through divergent mechanisms.

Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene <i>DNM1L</i>, affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological <i>DNM1L</i> variants impair mitochondrial network maintenance by divergent mechanisms.