Septins are a conserved family of cytoskeletal proteins implicated in a wide array of cellular functions including cell cycle regulation, ciliogenesis, and cell motility. Two of the most basic functions of septins seems to be compartmentalization of membrane structures and protein scaffolding. Of the four cytoskeletal protein families that provide structure and organization to the cellular environment, septins are the most recently discovered and the least characterized. Like tubulin and actin, septins bind nucleotide (GTP/GDP) and assemble into filaments via interactions between nucleotide-binding domains. Like the intermediate filaments, septins contain coiled-coils essential for the formation of higher-order assemblies. What sets septins apart is their striking structural plasticity. Septin subunits form apolar heteromeric complexes, which self-assemble into filaments, paired filaments, rings, and gauzes. My work has sought to answer three questions about septin plasticity and protein scaffolding. How are distinct septin subunits arranged in heteromeric complexes? How does the arrangement and composition of subunits relate to distinct assembly states and cellular functions? How do the scaffolding properties of septins regulate cellular processes? Using electron microscopy and biochemical techniques to study the structure and function of the mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) in the budding yeast Saccharomyces cerevisiae, I have found that Shs1 substitutes for Cdc11 at the terminal position in septin octamers and promotes the formation of septin rings, instead of the paired filaments formed by Cdc11-capped octamers. The rings observed in vitro are similar in size as those found in vivo, and Shs1 is essential for the robust formation of the ring-like septin collar at the site of cytokinesis. In addition to subunit exchange, I provide evidence that phosphorylation regulates septin self-assembly. Different phosphomimetic mutations of Shs1 either prevent ring assembly or promote the formation of an alternate assembly, septin gauzes. The implications of these results are discussed. I also studied the molecular organization and self-assembly properties of Spr3 and Spr28, two septins expressed specifically during meiosis and sporulation, an alternate developmental pathway induced by nutrient starvation. I also examined the mechanism by which septins serve as a protein scaffold for the cell-cycle regulator Hsl1, an AMPK protein kinase that appears to act as sensor for septin assembly.
This repository contains 2D morphology measurements from timelapse microscopy data of two interfertile Chlamydomonas algal species. The protocol to generate this data is described in the associated publication, "Phenotypic differences between interfertile Chlamydomonas species", and summarized here. Cells were collected from agar plates and suspended in water, then left to sit overnight to encourage gamete formation. During this time, non-motile cells settled, allowing for the enrichment of motile cells in the supernatant. These enriched cells were then loaded onto agar microchambers (100 micron diameter and 40 micron depth) for imaging. We collected videos on a Nikon Ti2-E microscope equipped with a Photometrics Kinetix digital scMos camera. We performed differential interference contrast (DIC) imaging using a Plan Apo 10× 0.45 Air objective. We collected videos with a 5.1 ms exposure with acquisition every 50 ms for three minutes. We placed a red light filter [IR longpass, 610 nm (ThorLabs)] in the light path to maintain swimming behavior of cells. The procedure was standardized and repeated four times to ensure consistency. Measurements collected with Cellprofiler of timelapse data of C. reinhardtii or C. smithii cells in agar microchamber wells are shared here. Reference Essock-Burns T, Garcia III G, MacQuarrie CD, Mets DG, York R. (2023). Phenotypic differences between interfertile Chlamydomonas species Notes Directory and subdirectories containing csv files of measurements of algal cells segmented from images.Directory structure: experiments_csv/{experiment}/{video_length}/objects/{species}/{microchamber AKA "pool ID"}/measurements/measurementschlamy.csv "Cr" indicates Chlamydomonas reinhardtii "Cs" indicates Chlamydomonas smithii
ABSTRACT Diverse human ciliopathies, including nephronophthisis (NPHP), Meckel syndrome (MKS) and Joubert syndrome (JBTS), can be caused by mutations affecting components of the transition zone, a ciliary domain near its base. The transition zone controls the protein composition of the ciliary membrane, but how it does so is unclear. To better understand the transition zone and its connection to ciliopathies, we defined the arrangement of key proteins in the transition zone using two-color stochastic optical reconstruction microscopy (STORM). This mapping revealed that NPHP and MKS complex components form nested rings comprised of nine-fold doublets. The NPHP complex component RPGRIP1L forms a smaller diameter transition zone ring within the MKS complex rings. JBTS-associated mutations in RPGRIP1L disrupt the architecture of the MKS and NPHP rings, revealing that vertebrate RPGRIP1L has a key role in organizing transition zone architecture. JBTS-associated mutations in TCTN2 , encoding an MKS complex component, also displace proteins of the MKS and NPHP complexes from the transition zone, revealing that RPGRIP1L and TCTN2 have interdependent roles in organizing transition zone architecture. To understand how altered transition zone architecture affects developmental signaling, we examined the localization of the Hedgehog pathway component SMO in human fibroblasts derived from JBTS-affected individuals. We found that diverse ciliary proteins, including SMO, accumulate at the transition zone in wild type cells, suggesting that the transition zone is a way station for proteins entering and exiting the cilium. JBTS-associated mutations in RPGRIP1L disrupt SMO accumulation at the transition zone and the ciliary localization of SMO. We propose that the disruption of transition zone architecture in JBTS leads to a failure of SMO to accumulate at the transition zone, disrupting developmental signaling in JBTS.
This repository contains 2D morphology measurements from timelapse microscopy data of two interfertile Chlamydomonas algal species. The protocol to generate this data is described in the associated publication, "Phenotypic differences between interfertile Chlamydomonas species", and summarized here. Cells were collected from agar plates and suspended in water, then left to sit overnight to encourage gamete formation. During this time, non-motile cells settled, allowing for the enrichment of motile cells in the supernatant. These enriched cells were then loaded onto agar microchambers (100 micron diameter and 40 micron depth) for imaging. We collected videos on a Nikon Ti2-E microscope equipped with a Photometrics Kinetix digital scMos camera. We performed differential interference contrast (DIC) imaging using a Plan Apo 10× 0.45 Air objective. We collected videos with a 5.1 ms exposure with acquisition every 50 ms for three minutes. We placed a red light filter [IR longpass, 610 nm (ThorLabs)] in the light path to maintain swimming behavior of cells. The procedure was standardized and repeated four times to ensure consistency. Measurements collected with Cellprofiler of timelapse data of C. reinhardtii or C. smithii cells in agar microchamber wells are shared here. Reference Essock-Burns T, Garcia III G, MacQuarrie CD, Mets DG, York R. (2023). Phenotypic differences between interfertile Chlamydomonas species Notes Directory and subdirectories containing csv files of measurements of algal cells segmented from images.Directory structure: experiments_csv/{experiment}/{video_length}/objects/{species}/{microchamber AKA "pool ID"}/measurements/measurementschlamy.csv "Cr" indicates Chlamydomonas reinhardtii "Cs" indicates Chlamydomonas smithii
Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.
Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.
