JCS snapshots: the cilia and flagella edition

Journal of Cell Science recently published a Special Issue on Cilia and Flagella: from Basic Biology to Disease, edited by Pleasantine Mill and Lotte Pedersen. We invited the authors of the research published in this issue to produce JCS snapshots, short videos highlighting the key findings from their papers. Below, you can find the snapshots from Rudolfo Karl and colleagues; Alyssa Long and colleagues; Melis Dalbay and colleagues; Laurence Wilson & Martin Bees; and Natalie Mosqueda and colleagues.

Renal tissue-resident macrophages promote cystogenesis in early polycystic kidney disease
Rudolfo Karl, Arsila Palliyulla Kariat Ashraf, Maria Francesca Viola, Katharina Hopp, Elvira Mass, Dagmar Wachten

Speaker: Rudolfo Karl

Autosomal-dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by mutations in PKD1 or PKD2, which drive cystogenesis in renal epithelial cells. Immune cells, particularly macrophages, contribute to disease progression, yet their role remains incompletely understood. Here, we performed an in-depth analysis of renal macrophage ontogeny and phenotype and investigated their function in an ADPKD mouse model (Pkd1^RC/RC) with adult onset and slow disease progression. We demonstrate that the numbers of tissue-resident macrophages were already increased before cyst formation. Using a flow cytometry screening panel, we further characterized the tissue-resident macrophage populations using surface markers and identified a novel marker that shows the potential to determine macrophage remodeling at different disease stages. To reveal the cellular interaction of tissue-resident macrophages and renal epithelial cells in further detail, we established a 3D co-culture system, demonstrating that tissue-resident macrophages from Pkd1^RC/RC mice, isolated at a stage before cysts were observed, already showed enhanced cystogenesis in vitro. These findings underscore the crucial role of tissue-resident macrophages in ADPKD and suggest targeting epithelial cell–macrophage interactions as a promising therapeutic avenue.

ARL13B-Cerulean rescues Arl13b-null mouse from embryonic lethality and reveals a role for ARL13B in spermatogenesis
Alyssa B. Long, Isabella M. Wilson, Tiffany T. Terry, Robert E. Van Sciver, Tamara Caspary

Speaker: Alyssa Long

ARL13B is a regulatory GTPase enriched in cilia, making it a popular marker for this organelle. Arl13b^hnn/hnn mice lack ARL13B expression, die during mid-gestation, and exhibit defects in ciliogenesis. The R26Arl13b-Fucci2aR biosensor mouse line directs the expression of fluorescently tagged full-length Arl13b cDNA upon Cre recombination. To determine whether constitutive, ubiquitous expression of Cerulean-tagged ARL13B (ARL13B-Cerulean) can replace endogenous gene expression, we generated Arl13b^hnn/hnn animals expressing ARL13B-Cerulean. We show that Arl13b^hnn/hnn;Arl13b-Cerulean mice survive to adulthood with no obvious physical or behavioral defects, indicating that the fluorescently tagged protein can functionally replace the endogenous protein during development. However, we observed that rescued males failed to sire offspring, revealing a role for ARL13B in spermatogenesis. This work shows that the R26Arl13b-Fucci2aR mouse contains an inducible allele of Arl13b capable of functioning in most tissues and biological processes.

Characterisation of a primary ciliary dyskinesia model generated from BMI1-transduced basal epithelial cells
Melis T. Dalbay, Eriomina Shahaj, Ileana Guerrini, Dani Do Hyang Lee, Anna Straatman-Iwanowska, Hannah M. Mitchison, Deborah L. Baines, Robert A. Hirst, Claire Hogg, Christopher O’Callaghan, Stephen L. Hart

Speaker: Melis Dalbay

Primary ciliary dyskinesia (PCD) is a rare genetic respiratory disorder caused by a reduction in cilia number or cilia dysmotility. Cilia dysmotility leads to breathing difficulties, concurrent infections and severe lung damage if not treated, with no therapies currently available. Improved airway epithelial cell models that mimic the disease phenotype are required for development of new therapeutics, as current models have limited potential of self-renewal in vitro. Here, we describe a human PCD cell model created by lentiviral transduction of airway basal epithelial cells with the BMI1 gene, a regulator of senescence. We report that the cells retain their proliferation and differentiation capacity for at least 19 passages and recapitulate the disease phenotype with immotile cilia lacking DNAH5 and other outer dynein arm proteins. Characterisation of the ion transport properties of these PCD cells grown at an air–liquid interface showed lower activity of the Na⁺ channel ENaC and enhanced CFTR activity compared to non-PCD cells, which might be linked to ciliary immotility. Our study provides a robust PCD model for therapeutic studies, opening new avenues to investigate the molecular mechanisms of this disease.

Asymmetries in the three-dimensional beat of Chlamydomonas reinhardtii flagella revealed by holographic microscopy
Laurence G. Wilson, Martin A. Bees

Speaker: Laurence Wilson

We present the first three-dimensional time-resolved imaging of the Chlamydomonas reinhardtii flagellar waveform. This freshwater alga is a model system for eukaryotic flagella that allow cells to move and pump fluid. During the power stroke, the flagella show rotational symmetry about the centre line of the cell, but during the recovery stroke they display mirror symmetry about the same axis. Furthermore, and in contrast to the usual assumptions about beat planarity, we show a subtle rotational motion of the flagella at the initiation of the power stroke, which is mechanically rectified into a quasi-planar mode. We apply resistive force theory to infer the swimming speed and rotational speed of the cells, when a force-free configuration is approximated using a cell on a micropipette, showing good agreement with experimental results on freely swimming cells.

Importin α regulates ciliogenesis and cilia length with implications for Xenopus nephrogenesis
Natalie Mosqueda, Patrick James Sutton, Christopher W. Brownlee

Speaker: Natalie Mosqueda

Cilia are microtubule-based organelles that are essential for a wide range of biological processes ranging from facilitating fluid flow to transducing developmental and growth signals. Defects in cilia structure or function can lead to ciliopathies. Ciliogenesis and cilia length regulation depend on protein transport to the ciliary base, but the underlying molecular mechanisms remain unclear. Here, we identify that the nuclear adapter protein importin α-1 (encoded by KPNA2 in humans; hereafter importin α) has conserved localization in human epithelial primary cilia and Xenopus laevis epidermal multiciliated cells. We find that importin α regulates both ciliogenesis and cilia length maintenance in a way that is dependent on both its localization to the membrane via palmitoylation and its presence in the cytoplasm when not palmitoylated. In addition, we identify key ciliary proteins, CEP164 and ARL13B, as candidate binding partners of importin α through their nuclear localization sequence (NLS) and the requirement of this binding interaction for proper ciliogenesis and cilia length. Disruption of importin α palmitoylation in X. laevis causes defects in nephrogenesis, which are rescued by forced membrane localization of importin α. These findings reveal a previously unrecognized role for importin α in cilia biology and advances understanding of congenital kidney diseases.

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