ABSTRACTThe inter-cellular prion-like propagation of α-synuclein aggregation is emerging as an important mechanism driving the progression of neurodegenerative diseases including Parkinson’s disease and multiple system atrophy (MSA). To discover therapeutic strategies reducing the spread of α-synuclein aggregation, we performed a genome-wide CRISPR interference screen in a human cell-based model. We discovered that inhibiting PIKfyve dramatically reduced α-synuclein aggregation induced with both recombinant α-synuclein fibrils and fibrils isolated from MSA patient brain. While PIKfyve inhibition did not affect fibril uptake or α-synuclein clearance or secretion, it reduced α-synuclein trafficking from the early endosome to the lysosome, thereby limiting fibril escape from the lysosome and reducing the amount of fibrils that reach cytosolic α-synuclein to induce aggregation. These findings point to the endolysosomal transport of fibrils as a critical step in the propagation of α-synuclein aggregation and a potential therapeutic target.

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments that have the same helical path but bind the membrane with different interfaces. Higher-resolution cryo-EM of filaments bound to helical bicelles confirms that ESCRT-III filaments can interact with the membrane through a previously undescribed interface. Mathematical modeling demonstrates that the interface described above is key to the mechanical stability of helical membrane tubes and helps infer the rigidity of the described protein filaments. Altogether, our results suggest that the interactions between ESCRT-III filaments and the membrane could proceed through multiple interfaces, to provide assembly on membranes with various shapes, or adapt the orientation of the filaments towards the membrane during membrane remodeling.

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2–4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline–arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8–13 and chromatin organization14–17. Following cell division, phase separation of the transmembrane adaptor LEM2 ensures that the ESCRT machinery remodels microtubules and seals the nuclear envelope.

Author(s): Johnson, Isabel Emily | Advisor(s): Frost, Adam | Abstract: Membranes are fundamental to cellular life. They define the cellular border and establish biochemically specialized subcellular compartments. As cell grow, change, and divide, cellular membranes are remodeled accordingly: undergoing membrane fission and fusion to maintain cellular architecture. For example, the nuclear membrane that organizes and protects DNA is remodeled during cell division. In cells that undergo “open mitosis”, the nuclear membrane is fully disassembled to allow chromosomes to be segregated by the spindle apparatus and reassembled following chromosome segregation. Understanding how cells remodel nuclear membranes to rebuild nuclei with every cell division is critical to understanding the core principals of membrane biology that underlie cellular life. In my dissertation, I describe how a membrane fusion complex is organized to seal holes in the reforming nuclear envelope that are occupied by spindle microtubules. Using biochemical approaches, I find that an inner nuclear membrane protein, called LEM2, uses multivalent, low-affinity binding interactions to condense in a liquid-like phase at the junction of the nuclear envelope, chromatin, and residual spindle microtubules. There, LEM2 activates the ESCRT protein, CHMP7, in a looping copolymer. Together, the fluid LEM2 toroid and structured LEM2-CHMP7 copolymer collaborate to serve as a molecular “O-ring” for early nuclear sealing. Furthermore, the LEM2-CHMP7 ring serves as a foundation for the membrane-remodeling ESCRT-III complex to execute coupled spindle disassembly and membrane fusion. This work establishes a new paradigm in membrane organization, in which a liquid-like protein phase can function as both a barrier within a discontinuous membrane and a scaffold for membrane fusion factors.

At mitotic exit, microtubule arrays are dismantled in concert with the reformation of the nuclear envelope. We show how the inner nuclear membrane protein, LEM2, exploits liquid-liquid phase separation to direct microtubule remodeling and nuclear envelope sealing via the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway. LEM2 tethers membrane to chromatin disks through direct binding between its LEM motif and the chromatin-associated barrier-to-autointegration factor (BAF). Concurrently, a low-complexity domain within LEM2 undergoes liquid-liquid phase separation to coat spindle microtubule bundles. Spatially restricted, LEM2’s winged helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7. Together LEM2 and CHMP7 copolymerize around microtubule bundles to form a molecular “O-ring” that promotes nuclear compartmentalization and initiates downstream ESCRT factor recruitment. These results demonstrate how multivalent interactions of a transmembrane protein, including those that mediate phase separation, coordinate localized ESCRT polymerization, mitotic spindle disassembly, and membrane fusion. Defects in this pathway compromise spindle disassembly, nuclear integrity, and genome stability.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Coenzyme Q (Q) is an essential lipid best known for its role as an electron carrier in the electron transport chain. Although required for human health, Q biosynthesis is not completely understood and the functions of many Q biosynthetic proteins remain unclear. The atypical mitochondrial kinase, ADCK3, is a Q biosynthetic protein of unknown function belonging to the UbiB protein kinase‐like family. Here, we determine a crystal structure of ADCK3, the first crystal structure of a UbiB protein. We characterize the unique alanine‐rich nucleotide binding loop that dictates an unusual binding preference for ADP over ATP. We find that this preference can be reversed with a single alanine to glycine mutation, which also enables autophosphorylation while inhibiting Q biosynthesis in vivo. Furthermore, we identify an N‐terminal domain that blocks the substrate binding pocket—another inhibitory feature. This work serves as a foundation for further structure‐function analyses aimed at defining the role of ADCK3 in Q biosynthesis as part of a larger effort to treat human Q deficiency.

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

The UbiB family of kinases represents an estimated 25% of the protein kinase‐like (PKL) family members in the microbial kingdom, and members of this family are required for the biosynthesis of coenzyme Q from E. coli to humans. We present the first structure of a UbiB family member, the human mitochondrial kinase ADCK3, which is mutated in a human cerebellar ataxia (ARCA2) associated with coenzyme Q deficiency. The structure of ADCK3 reveals that the signature, invariant KxGQ motif of UbiB kinases interacts directly with the kinase active site, in close proximity to residues important for nucleotide binding. Differential scanning fluorimetry (DSF) and deuterium exchange mass spectrometry were used to confirm that this motif is important for proper nucleotide binding, and to establish that ADP binding has a global stabilizing effect on the kinase. Last, mapping ADCK3 patient mutations onto our structure suggests how these mutations can destabilize the kinase. We purified and analyzed the thermal stability of eight such ARCA2 patient mutations using DSF and found that all but three of these proteins are less stable than wild type ADCK3. Our findings form the foundation for studying the enzymatic activity of the UbiB family and for investigating how disruptions in this kinase can give rise to a human neurodegenerative disorder.Grant Funding Source: A.G.R. was supported by NIH Chemistry‐Biology Interface Training Grant NIGMS T32 GM008505, D.J.P. was supported by NIH Grant U01 GM94622 (Protein Structure Initiative: Biology, Mitochondrial Protein Partnership) and a Searle Scholars Award