Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 6, Pages E1221-E1228Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1715915115
Keywords
ciliopathy; cilia; genetics; preassembly; primary ciliary dyskinesia
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Funding
- American Thoracic Society Foundation/Primary Ciliary Dyskinesia Foundation/Kovler Family Foundation
- NIH [HL128370]
- Washington University Center for Cellular Imaging award through a grant from The Children's Discovery Institute of Washington University
- St. Louis Children's Hospital [CDI-CORE-2015-505]
- Foundation for Barnes-Jewish Hospital [3770]
- Foundation for Barnes-Jewish Hospital
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Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Forster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.
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