Synopsis
First discovered in the early 1960’s, the primary cilium remained an “abortive structure with no function” for four decades. Fast forward to 2006 and the term “signaling antenna” is coined to describe primary cilia. This 180-degree shift in perception was fueled by two major advances: First, the ciliary hypothesis of polycystic kidney disease spread to the recognition of ciliary dysfunction as the pathological basis for the ciliopathies, a broad class of disorders characterized by obesity, cystic kidneys, retinal degeneration, skeletal malformations and brain anomalies. Second, Hedgehog signaling, a major developmental signaling pathway responsible for patterning the neural tube was shown to require cilia for its signal transduction. The cilium has now emerged as a vessel that transforms signaling inputs into processed information for the rest of the cell by dynamically concentrating signaling molecules within a specialized physicochemical environment.
Motivated by the tremendous importance of cilia in human health and disease and in signal transduction, our work aims to understand the basic principles of trafficking into and out of cilia and how the ciliary dynamics of signaling molecules affect signaling outputs. Historically, our lab pioneered the use of biochemical purifications of ciliopathy proteins to discover the BBSome, a protein complex with structural similarities to the coat complexes COPI, COPII and clathrin. In subsequent years, we developed single-molecule tracking, photokinetic assays and in vitro reconstitution to mechanistically dissect BBSome-mediated transport. Most recently, we leveraged nascent advances in proximity labeling to catalogue the protein content of cilia and profile alterations in the ciliary proteome that arise from ciliopathy mutations. Our combined expertise in proteomics, biochemistry and imaging places us in a unique position to answer the central questions in ciliary biology.
Motivated by the tremendous importance of cilia in human health and disease and in signal transduction, our work aims to understand the basic principles of trafficking into and out of cilia and how the ciliary dynamics of signaling molecules affect signaling outputs. Historically, our lab pioneered the use of biochemical purifications of ciliopathy proteins to discover the BBSome, a protein complex with structural similarities to the coat complexes COPI, COPII and clathrin. In subsequent years, we developed single-molecule tracking, photokinetic assays and in vitro reconstitution to mechanistically dissect BBSome-mediated transport. Most recently, we leveraged nascent advances in proximity labeling to catalogue the protein content of cilia and profile alterations in the ciliary proteome that arise from ciliopathy mutations. Our combined expertise in proteomics, biochemistry and imaging places us in a unique position to answer the central questions in ciliary biology.
Proteomics of primary cilia
The full extent of signaling molecules organized by cilia remains unknown. Since cilia purification is not feasible in mammalian cells, we set out to identify ciliary proteins by targeting the proximity biotinylation enzyme APEX to cilia and capturing biotinylated proteins. The development of the cilia-APEX technology led to the identification of novel signaling axes in cilia. These include the AMP-regulated protein kinase (AMPK), pointing to a possible role of cilia in sensing energetic cues, and the cAMP-dependent protein kinase (PKA), which led us to show that Hedgehog signals are transduced by intra-ciliary cAMP and PKA. It is tempting to speculate that cilia regulate second messenger concentrations separately from the rest of the cell, thus affording localization-dependent regulation to various signaling pathways.
The advent of straightforward primary cilia proteomics constitutes a breakthrough in the field of primary cilia biology. First, the sensitivity of cilia-APEX surpasses traditional methods of immunodetection. We have uncovered proteins that rapidly traverse cilia and remain undetectable by immunohistochemitry yet play important roles inside cilia. Second, the minute-scale temporal resolution of cilia-APEX shall enable a characterization of the changes in the ciliary proteome that take place during the course of Hedgehog signaling and other ciliary signaling pathways. Finally, profiling the genotype-dependent alterations of the ciliary proteome will help uncover the molecular etiology underlying each ciliopathy, offering hope for novel mechanistic insights into ciliary biology.
The advent of straightforward primary cilia proteomics constitutes a breakthrough in the field of primary cilia biology. First, the sensitivity of cilia-APEX surpasses traditional methods of immunodetection. We have uncovered proteins that rapidly traverse cilia and remain undetectable by immunohistochemitry yet play important roles inside cilia. Second, the minute-scale temporal resolution of cilia-APEX shall enable a characterization of the changes in the ciliary proteome that take place during the course of Hedgehog signaling and other ciliary signaling pathways. Finally, profiling the genotype-dependent alterations of the ciliary proteome will help uncover the molecular etiology underlying each ciliopathy, offering hope for novel mechanistic insights into ciliary biology.
Barriers of the cilium
From Anderson RGW (1972). JCB 54:246–265.
With a volume of 0.3 femtoliters, the cilium encloses one ten-thousandth of the cytoplasmic volume. It is inferred that relocating 100 copies of a molecule from the cytoplasm to the cilium will increase its concentration form 50 pM to 0.5 μM. To achieve such tremendous concentration against their concentration gradient, proteins rely on diffusion barriers that separate the ciliary membrane and its soluble interior from the rest of the cell and make the cilium the sole surface-exposed compartment of the cell. We uncovered the existence of the soluble diffusion barrier using a classical cell biology approach of semi-permeabilization that perforates the plasma membrane while leaving the ciliary membrane and ciliary pore complex intact. The resulting cell ghosts enabled a powerful mechanistic dissection of protein diffusion into and inside cilia. We found that soluble proteins smaller than 75 kDa can passively enter cilia and that the permeability barrier at the base of cilia is mechanistically distinct from those operating at the ciliary pore complex and at the axon initial segment. For large proteins, the mechanisms of selective entry into cilia remain largely uncharacterized. Armed with a powerful semi-permeabilized cell system, we aim to dissect the mechanisms of active entry into cilia.
Regulated ciliary trafficking
A hallmark of ciliary signaling pathways is the dynamic redistribution of signaling molecules in and out of cilia upon pathway engagement. Considering genetics as a means to initiate the molecular dissection of a novel process, we leveraged the vast numbers of molecular entry points provided by human genetics and applied tandem affinity purification and mass spectrometry to identify complexes of ciliopathy proteins. While the identification of the BBSome as a stable complex of eight Bardet-Biedl proteins was relatively straightforward, deciphering the precise molecular activity of a novel complex required the development of innovative assays and led to surprising and unexpected research directions. A combination of secondary structure prediction, biochemical reconstitution with pure proteins, liposome-based assays and trafficking assays led to the current model of the BBSome as a coat complex that traffics membrane proteins into and out of cilia, a model that has influence subsequent studies finding a role for the BBSome in the ciliary trafficking of numerous signaling receptors.
Where, when and how BBSome-mediated trafficking is initiated remains a matter of debate and even the direction of BBSome-mediated transport is still an open question. To better understand how molecules are trafficked into and out of the cilium, we have developed a battery of assays in the last few years:
Where, when and how BBSome-mediated trafficking is initiated remains a matter of debate and even the direction of BBSome-mediated transport is still an open question. To better understand how molecules are trafficked into and out of the cilium, we have developed a battery of assays in the last few years:
- Single molecule tracking of signaling receptors. By tracking single GPCRs inside cilia, we found that diffusion was the predominant mode of signaling receptor movement inside cilia. This technique is now being applied to visualize how signaling alters the dynamics of signaling receptors moving into, within and out of cilia.
- Photokinetic measurements of bulk entry and exit rates. The application of superbright fluorescent proteins to ciliary trafficking complexes has made it possible to apply Fluorescence recovery after photobleaching (FRAP) and Fluorescent loss in photobleaching (FLIP) and molecular counting to measure absolute rates of ciliary entry and exit. These incisive assays made it possible to show that the BBS protein IFT27/RABL4/BBS19 primes the small GTPase ARL6/BBS3 within cilia to trigger exit of BBSome and cargoes out of cilia.
- We are currently developing methods to better approximate the expression levels of signaling receptors inside cilia. Those are present at less than 1000 copies per cilia and current expression methods have proven inadequate to recapitulate their expression levels and ciliary dynamics.
Microtubule acetylation
In 2010, we made the serendipitous discovery of the long-sought tubulin acetyltransferase TAT1/MEC-17. Unlike any other microtubule-associated protein or microtubule modifying enzyme characterized to date, TAT1 functions inside the lumen of hollow microtubules. To this date, the molecular consequences of tubulin acetylation remain enigmatic. At the cellular level, we found that tubulin acetylation is required for contact inhibition of proliferation, likely acting through the Hippo pathway. A current project aims to understand how acetylation modifies basic biophysical properties of microtubules, utilizing a combination of FRET assays, microfabrication and mechanical perturbations.
updated 2/4/2016
