Research Overview
Research in our laboratory focuses on furthering our understanding of nuclear compartmentalization. To this end, we are developing methodology to study the interplay of membrane dynamics, liquid-liquid phase separation and protein quality control. Our ultimate goal is to understand how defects in these processes are connected to human disease.
Recent Publications
Check out our recent paper in Nature Cell Biology reporting the discovery of a novel chaperone complex regulating the condensation of nucleoporins during nuclear pore assembly. A defect in this process causes the severe movement disorder Primary Dystonia.
See also two highlighting articles by Kirstein and DiFonzo & Zech.
- Torsin ATPases
- Protein Quality Control in the Nuclear Envelope
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4TOR KO projection.avi
Role of Torsin ATPases at the nuclear envelope and their relation to DYT1 dystonia
Torsins require regulatory cofactors, LAP1 and LULL1, to hydrolyze ATP. This is achieved through an active site complementation in which either cofactor contributes an essential arginine residue (termed arginine finger) that projects into the ATP binding pocket of an adjacent Torsin subunit. The structure shown depicts the association of LULL1 (pink) with a TorsinA Walker B mutant (blue). In the zoomed in view of the nucleotide binding pocket, key residues of the Walker A (K108) and Walker B (E171Q) motifs, as well as the two sensor motifs residing in TorsinA and the arginine finger of LULL1 are highlighted. PNS: perinuclear space, ER:endoplasmic reticulum, NP: nucleoplasm.
AAA+ (ATPases associated with various cellular activities) ATPases are a molecular machines that use the energy of ATP binding and hydrolysis to perform cellular work. These manifest as numerous energy-dependent proteases, helicases, unfoldases/chaperones, and motor proteins within the cell. Although AAA+ ATPases are ubiquitously present throughout the cell, Torsin ATPases (Torsins) including TorsinA and its three human orthologs (TorsinB, Torsin2A, and Torsin3A) are the only known members of this superfamily to localize to the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). Moreover, unlike other related AAA+ ATPases, Torsins require NE and ER specific cofactors, LAP1 and LULL1, to hydrolyze ATP. Studying Torsin ATPases has significant medical implications as a deletion of a single amino acid residue (E303) from TorsinA has been identified as the principle cause of DYT1 dystonia, a highly debilitating condition characterized by sustained muscle contractions and involuntary twisting.
Structural and biochemical insight into Torsin activity
Proposed models for Torsin-cofactor assembly dynamics. Torsin oligomeric assemblies (green) occupy either a planar (A) or stack spiral (B) conformation. Upon cofactor (purple) binding, ATP hydrolysis is stimulated which in turn triggering the disassembly of Torsin rings. Figure adapted from Chase et al. 2017.
Our laboratory was the first to establish a functional in vitro system to study Torsin ATPases, and we have since made a number of exciting and unexpected discoveries. We demonstrated that Torsin ATPases are outliers of the AAA+ superfamily of ATPases in the human genome in that they lack significant basal ATPase activity and that their catalytic activity is strictly reliant on LAP1 and LULL1, two accessory cofactors that accelerate the hydrolysis step by several orders of magnitude (P.N.A.S. 110(17):E1545-54) by virtue of an active site complementation mechanism (P.N.A.S. 111(45):E4822-3). Importantly, we found that this activation mechanism is offset by disease-causing mutations, which is the first direct demonstration of a loss-of-function mechanism for DYT1 Dystonia.
We have since shown that Torsins form homotypic oligomers in the presence of ATP, which are rapidly disassembled upon cofactor engagement and ATP hydrolysis. We propose a model in which Torsin’s distinctively localizing cofactors act as modulators of Torsin oligomeric assemblies providing a means of regulating these essential ATPases (Chase et al. 2017,Chase et al. 2017b).
Novel role of Torsin ATPases in nuclear pore assembly
Nuclear envelope (NE) blebbing is the phenotypic hallmark resulting from Torsin manipulation. (A) EM micrograph (left) and false color EM tomogram (right) demonstrating NE blebbing. The arrow highlights the electron density at the neck region. (B) EM micrograph of NE blebs with immunogold labeling using Mab414, an antibody recognizing several FG-rich nucleoporins. This subset of nups localize to the neck of blebs indicative of the presence of a NPC assembly intermediate. Figure adapted from Laudermilch et al. 2016.
Recently, our group has engineered an altogether Torsin-deficient cell line through CRISPR genome editing technology that recapitulates the aberrant NE structures that are the phenotypic hallmark resulting from Torsin manipulation in a wide variety of metazoans (Laudermilch et al.). More recently, we pioneered a live cell imaging platform to show that NE blebs represent stalled intermediates in nuclear pore biogenesis (for more details please see our recent publication on bioRxiv (https://doi.org/10.1101/821835).
Current research in our laboratory is focused on examining the specific cellular and mechanistic roles of Torsin-cofactor complexes (see Chase et al. 2017 for a perspective article), with specific emphasis on nuclear envelope dynamics and nuclear pore biogenesis (Laudermilch et al., https://doi.org/10.1101/821835).By employing CRISPR/Cas9-based genetic tools, classical biochemical fractionation, mass spectrometry, in vitro reconstitution and confocal as well as lattice light sheet microscopy, we seek to arrive at a comprehensive understanding of the Torsin system both on a molecular and a cell biological level.