Biology subjects

Gustafson, J. A.

Publications and source records attributed to Gustafson, J. A..

5 recordsLinked to original sources

bioRxiv2025

Pathogenic DVL frameshifting variants in Robinow syndrome disrupt WNT signaling and cellular dynamics

Robinow syndrome (RS) is a genetically heterogeneous rare disorder involving six genes in the WNT/planar cell polarity (PCP) signaling pathway. Frameshifting variants in DVL genes that introduce a novel basic C-terminus are a common cause of autosomal dominant RS, accounting for [~]33% of individuals without ROR2 variants. Here, we review ClinVar and literature variants affecting DVL paralogs resulting in RS and investigate the cellular effects of pathogenic DVL frameshift variants with mutant tails replacing at least 82 amino acids. In silico analysis of the new C-termini revealed altered intrinsically disordered regions (IDRs), charge distribution, and predicted protein structures. To explore potential altered biological mechanisms caused by novel C-termini, we generated wild-type (WT), frameshift, and truncated constructs of DVL1-3, and analyzed their behavior in a transfection-based in vitro systems. DVL proteins normally polymerize into cytoplasmic puncta that redistribute upon WNT stimulation. Immunocytochemistry showed that mutant DVL proteins failed to change their localization in response to WNT ligands, in contrast to WT alleles--a consistent observation across all three DVLs. In line with this, TOPFlash reporter assays demonstrated that mutant DVL1 and DVL3 failed to activate canonical WNT signaling, while WT proteins induced strong activation. Additionally, the mutant C-terminal tail interfered with CSNK1E-induced phosphorylation, offering a potential mechanism underlying the impaired WNT response. Our results provide further understanding of the cellular consequences of pathogenic DVL frameshifting variants and offers insights into the effects of such alleles on WNT signaling, and the perturbations thereof, that may lead to developmental phenotypes observed in RS.

genetics↗

bioRxiv2025

Principles and functional consequences of plasmid chromatinization in mammalian cells

Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, although chromatin plays an integral role in regulating the function of regulatory elements along the nuclear genome, our understanding of how, or whether, similar chromatin structures form on plasmids transfected into mammalian cells remains limited. We demonstrate that plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) can accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near single-nucleotide resolution. Application of plasmid Fiber-seq to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in a sequence-dependent organized manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. We show that the focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity within mammalian cells, and demonstrate that plasmids indeed are capable of recapitulating nuclear genome-encoded chromatin architectures, although not always. Finally, we demonstrate that combining plasmid Fiber-seq with high-throughput reporter assays can establish the molecular mechanisms underlying pathogenic non-coding variants, including disentangling the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Overall, our findings reveal the principles by which plasmid-based assays can be used for accurate fine-scale mapping of chromatin-dependent regulatory grammar.

genomics↗

bioRxiv2024

Personalization of Closed-Chain Shoulder Models Yields High Kinematic Accuracy for Multiple Motions

The shoulder joint complex is prone to musculoskeletal issues, such as rotator cuff-related pain, which affect two-thirds of adults and often result in suboptimal treatment outcomes. Current musculoskeletal models used to understand shoulder biomechanics are limited by challenges in personalization, inaccuracies in predicting joint and muscle loads, and an inability to simulate anatomically accurate motions. To address these deficiencies, we developed a novel, personalized modeling framework capable of calibrating subject-specific joint centers and functional axes for the shoulder complex. Leveraging in vivo biplane fluoroscopy data and the recent Joint Model Personalization Tool from the Neuromusculoskeletal Modeling Pipeline, we optimized joint parameters and body scale factors for shoulder models with varying degrees of freedom (DOFs). We initially created and tested open-chain scapula-only models (3DOF, 4DOF, and 5DOF) and found that increasing DOFs improved accuracy, with the 5 DOF model yielding the lowest marker distance errors (average = 0.8 mm, maximum = 5.2 mm) as compared to biplane fluorscopy data of the scapula across eight movement trials. We subsequently created closed-chain shoulder models incorporating scapula, clavicle, and humerus bodies. We found closed-chain shoulder models with 5 DOFs for the scapula achieved the highest accuracy (average = 0.9 mm, maximum = 5.7 mm) and showed consistent performance across subjects (n=3) in leave-one-out cross-validation tests (average marker distance errors = 1.0-1.4 mm). This framework minimizes errors in joint kinematics and provides a foundation for future models incorporating personalized musculature and advanced simulations, enhancing its potential clinical utility for rehabilitation and surgical planning.

bioengineering↗

bioRxiv2022

State-dependent protein-protein interactions mediating 4-1BB CAR Signaling

Cells rely on activity-dependent protein-protein interactions to convey biological signals, but the state-dependent interactome is notoriously cell-specific and undercharacterized1. In the case of chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to trigger the formation of protein complexes similar to those triggered by T cell receptor (TCR)-mediated signaling, but the number and type of protein-interaction-mediating binding domains differ between CARs and TCRs. Here, we performed co-immunoprecipitation mass spectrometry of a 2nd generation CD19-directed 4-1BB:zeta CAR (referred to as bb{zeta}CAR) and identified 67 proteins that increased their co-association after target engagement. We compared activity-induced TCR and CAR signalosomes using quantitative multiplex co-immunoprecipitation and showed that bb{zeta}CAR engagement leads to activation of two modules of protein interactions, one similar to TCR signaling that is more weakly engaged in bb{zeta}CAR vs. TCR, and one composed of TRAF signaling complexes that is not engaged by the TCR. Batch-to-batch and inter-individual variations in IL2 production correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers. One Sentence SummaryWe define a network of protein interactions engaged by chimeric antigen receptors following target binding, and show that the magnitude of network activation correlates with IL-2 secretion, a proxy measure for CAR T cell function.

immunology↗

bioRxiv2022

Interaction of surface topography and taper mismatch on head-stem modular junction contact mechanics during assembly in modern total hip replacement

Implant failure due to fretting corrosion at the head-stem modular junction is an increasing problem in modular total hip arthroplasty. The effect of varying microgroove topography on modular junction contact mechanics has not been well characterized. The aim of this work was to employ a novel, microgrooved finite element (FEA) model of the hip taper interface and assess the role of microgroove geometry and taper mismatch angle on the modular junction mechanics during assembly. A two-dimensional, axisymmetric FEA model was created using a modern 12/14 taper design of a CoCrMo femoral head taper and Ti6Al4V stem taper. Microgrooves were modelled at the contacting interface of the tapers and varied based on height and spacing measurements obtained from a repository of measured retrievals. Additionally, taper angular mismatch between the head and stem was varied to simulate proximal- and distal-locked engagement. Forty simulations were conducted to parametrically evaluate the effects of microgroove surface topography and angular mismatch on predicted contact area, contact pressure, and equivalent plastic strain. Multiple linear regression analysis was highly significant (p < 0.001; R2 > 0.74) for all outcome variables. The regression analysis identified microgroove geometry on the head taper to have the greatest influence on modular junction contact mechanics. Additionally, there was a significant second order relationship between both peak contact pressure (p < 0.001) and plastic strain (p < 0.001) with taper mismatch angle. These modeling techniques will be used to identify the implant parameters that maximize taper interference strength via large in-silico parametric studies.

bioengineering↗