Biology subjects

Robert, K. L.

Publications and source records attributed to Robert, K. L..

4 recordsLinked to original sources

bioRxiv2026

Ift43 Controls the Ciliary Levels of Gli2 and Gli3

Intraflagellar transport (IFT) drives the bidirectional movement of trains composed of IFT-A, IFT-B, and BBSome complexes that build and maintain cilia while supporting their signaling functions. Over evolution, IFT became integral to Hedgehog signaling by directing the dynamic movements of receptors and Gli transcription factors that fine-tune pathway output. The IFT-A complex contains six subunits, but the smallest, Ift43, remains poorly characterized and is absent from many ciliated species, suggesting specialized roles in signaling rather than core ciliogenesis. Here we show that loss of Ift43 in mice causes mid-gestation lethality with severe craniofacial defects, exencephaly, abdominal wall defects with exposed viscera, edema, and limb patterning defects. At the cellular level, Ift43 deficiency reduces both the number and length of cilia and blocks induction of Gli1 following pathway activation by the agonist SAG. Although Smoothened relocalizes to cilia normally, Ift43 mutants abnormally accumulate Gli2 and Gli3 at ciliary tips before stimulation and continue to generate repressor forms after activation. Conversely, Ift43 overexpression increases basal Gli2 cleavage, revealing an unanticipated role for Ift43 in regulating Gli processing. Together, these findings identify Ift43 as a key IFT-A component that links ciliary assembly to Hedgehog signal transduction and helps set the balance between Gli activator and repressor forms.

cell biology↗

bioRxiv2025

Loss of ADAMTS9 results in Nephronophthisis like polycystic kidneys by disrupting ciliogenesis and ECM dynamics

ADAMTS9 mutations cause the ciliopathies nephronophthisis and Joubert syndrome. Here we show that deletion of ADAMTS9 in the proximal nephron leads to polycystic kidney development in mice. In males, Adamts9 deletion cause kidneys to become highly cystic but remain small without undergoing enlargement, causing early postnatal lethality. Female mice on the other hand, develop cystic kidneys but progress slowly. ADAMTS9 deletion disrupted ciliogenesis by the loss of ciliary transition zone (TZ) protein TMEM67 cleavage, leading to loss of the MKS/B9 module - a key component of the ciliary gate. Functional analysis of all eight ciliopathy patient variants of ADAMTS9 identified to date, showed TMEM67 C-terminus failed to localize to the transition zone, thus disrupting a key regulatory mechanism in patient renal ciliogenesis. Modeling ADAMTS9-mediated TMEM67 cleavage utilizing our novel TMEM67-cleavage deficient mice revealed loss of TZ formation but not elevated canonical Wnt signaling as the underlying mechanism driving cystogenesis. We show that Adamts9 deletion leads to comparatively intense interstitial collagen deposition, which likely restricts kidney enlargement resulting in the characteristically small kidney phenotype in nephronophthisis and increased immune response. By comparative analysis of four interconnected polycystic kidney models in addition to Pkd1 and Pkd2 deleted kidneys, we identify differential collagen homeostasis is the principle determining factor deciphering cystic kidney size and type.

cell biology↗

bioRxiv2024

Two functional forms of the Meckel-Gruber syndrome protein TMEM67 generated by proteolytic cleavage by ADAMTS9 mediate Wnt signaling and ciliogenesis

TMEM67 mutations are the major cause of Meckel-Gruber syndrome. TMEM67 is involved in both ciliary transition zone assembly, and non-canonical Wnt signaling mediated by its extracellular domain. How TMEM67 performs these two separate functions is not known. We identify a novel cleavage motif in the extracellular domain of TMEM67 cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage regulates the abundance of two functional forms: A C-terminal portion which localizes to the ciliary transition zone regulating ciliogenesis, and a non- cleaved form which regulates Wnt signaling. By characterizing three TMEM67 ciliopathy patient variants within the cleavage motif utilizing mammalian cell culture and C. elegans, we show the cleavage motif is essential for cilia structure and function, highlighting its clinical significance. We generated a novel non-cleavable TMEM67 mouse model which develop severe ciliopathies phenocopying Tmem67-/- mice, but in contrast, undergo normal Wnt signaling, substantiating the existence of two functional forms of TMEM67.

cell biology↗

bioRxiv2022

Degradomic identification of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) as an ADAMTS9 and ADAMTS20 substrate

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix (ECM) proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative N-terminomics method, terminal amine isotopic labeling of substrates (TAILS) was used to compare parental and gene-edited cells and their medium to identify ADAMTS9 substrates. Among differentially abundant N-terminally labeled internal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease MT1-MMP, whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge i.e., between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by re-expression of ADAMTS9 and ADAMTS20, and was dependent on hinge O-glycosylation. Since MT1-MMP is a type I transmembrane protein, identification of an N-terminally labeled peptide in the medium suggested additional downstream cleavage sites in its ectodomain. Indeed, a C-terminally semi-tryptic MT1-MMP peptide with greater abundance in wild-type RPE-1 medium identified by a targeted search indicated a cleavage site in the hemopexin domain. Consistent with retention of MT1-MMP catalytic domain on the surface of gene-edited cells, pro-MMP2 activation, which requires cell-surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell-surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell-surface activity of MT1-MMP and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis. HighlightsO_LIADAMTS9-deficient RPE-1 cells have impaired substrate attachment C_LIO_LIADAMTS9 and ADAMTS20 release the MT1-MMP catalytic domain from the cell-surface C_LIO_LIIncreased cell-surface MT1-MMP increases pro-MMP2 activation and collagenolysis C_LIO_LIMT1-MMP knockdown restores substrate attachment of ADAMTS9-deficient RPE-1 cells. C_LI In BriefADAMTS9 and ADAMTS20 are homologous secreted proteases implicated in ECM proteolysis and ciliogenesis, but few relevant substrates of these proteases are currently known. Quantitative N-terminomics comparison of RPE-1 cells with ADAMTS9 inactivation and parental RPE-1 cells identified transmembrane protease MT1-MMP (MMP14) as a novel ADAMTS9 substrate. The resulting enhanced cell-surface MT1-MMP activity in the gene-edited cells contributes to their adhesion defect, but not lack of cilia. A key physiological function of ADAMTS9/20 may be to dampen cell-surface MT1-MMP activity.

biochemistry↗