Manu Prakash, Postdoctoral Faculty Sponsor
H-ABC- and dystonia-causing TUBB4A mutations show distinct pathogenic effects.
2022; 8 (10): eabj9229
Mutations in the brain-specific beta-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.
View details for DOI 10.1126/sciadv.abj9229
View details for PubMedID 35275727
Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.
Nature cell biology
The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.
View details for DOI 10.1038/s41556-021-00825-4
View details for PubMedID 35102268
Solid-State NMR Spectroscopy for Studying Microtubules and Microtubule-Associated Proteins
STRUCTURAL PROTEOMICS, 3 EDITION
2021; 2305: 193-201
In this chapter, we describe the preparatory and spectroscopic procedures for conducting solid-state NMR experiments on microtubules (MTs) obtained from human cells and their complexes with microtubule-associated proteins (MAPs). Next to labeling and functional assembly of MTs and MT-MAP complexes, we discuss solid-state NMR approaches, including fast MAS and hyperpolarization methods that can be used to examine these systems. Such studies can provide novel insight into the dynamic properties of MTs and MT-MAP complexes.
View details for DOI 10.1007/978-1-0716-1406-8_10
View details for Web of Science ID 000691017300011
View details for PubMedID 33950391