Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.

No abstract

Astrocytes are the most abundant glial cell in the central nervous system. In the brain specifically, astrocytes control or contribute to metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Astrocytes are well established as important players in understanding homeostatic brain function and disease, albeit, there are few methods to visualize or target these cells, greatly hindering our capability to fully characterize and study them. New therapeutic methods or tools to target astrocytes have the potential to combat neurodegenerative and neuropsychiatric conditions like depression, epilepsy, or Parkinson's. Each condition is characterized by either an imbalance in ion, neurotransmitter, or protein clearance— processes in the brain all modulated by astrocytes. Current methods utilizing genetic markers or fluorescent small molecules to target astrocytes suffer from a need for fixation, specificity concerns, gap junction diffusion, or an incapability for chemical modification. We have combated a few of these caveats by synthesizing an array of small molecule probes, recently demonstrating that we can deliver a variety of fluorophores to astrocytes. By leveraging organic chemistry to attach permanently positive, pyridinium‐like moieties to a variety of fluorescent small molecules, we have successfully labelled astrocytes without targeting neurons or other glia. Here, we explore the delivery of diverse small molecule cargo like transcriptional activators, calcium sensors, or drugs when modified with our astrocyte‐selective probes. Leveraging organic chemistry to attach permanently positive N‐heterocyclic amines to biologically interesting molecules will allow us to further probe astrocyte basic function and biology, all while informing the development of future tools in chemical biology. Here, we assay a modified small molecule calcium indicator and a doxycycline‐modified probe. The former will serve to mark activated astrocytes to characterize their response to various brain stimuli. Additionally, the calcium probe will help identify astrocytes that may be in direct communication with subsets of neurons. Painting a picture of the live calcium response in astrocytes could inform therapeutic development by revealing disease mechanisms in epilepsy or seizure, for example. Alternatively, the latter doxycycline probe will function as an astrocyte‐specific, transcription activator in the tetracycline‐inducible gene expression system. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. The doxycycline probe will be used to selectively activate transcription of genes of interest in astrocytes in order to control when and how the gene product is produced in a given model. We hope to employ these, and similar molecules therapeutically where current constitutive gene therapies for Parkinson's or glioblastoma, for example, could benefit from regulated gene expression. These probes demonstrate the diver...

Astrocytes are glial cells that tile the entire central nervous system. Within the brain, they control metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Targeting astrocytes using gene therapies could be an attractive therapeutic option for seizure, epilepsy, Parkinson’s or Alzheimer’s disease, where there exists an imbalance in ion, neurotransmitter, or protein clearance— processes all modulated by astrocytes. We have recently developed chemical tags that ferry small molecule cargo into astrocytes. Here, we adapt these astrocyte targeting tags to control transcription, with the ultimate goal of using them for regulated gene therapy. Essentially, we utilize a modified transcription activator, doxycycline, to turn on the tetracycline‐inducible gene expression (Tet‐On) system— obtaining temporal control over transcription in astrocytes. Astrocyte specificity is attained by attaching a permanently positive astrocyte targeting moiety via organic chemistry to the small molecule drug doxycycline, which retains its Tet‐On activating capabilities. Our current probes include doxycycline and 9‐cyanodoxycyline variants with appended targeting moieties to improve astrocyte targeting and reduce cell permeability, decreasing off‐target affects. Our first probe, doxycycline methyl pyridinium (doxyMP) can successfully activate transcription in primary astrocytes under the strong, mammalian cytomegalovirus (CMV) promoter, eliminating the need for a cell‐specific promoter. In classical gene therapies, constitutive systems suffer from off‐target effects or variable gene expression of cell‐specific promoters. Using an astrocyte‐targeted transcription activator, we eliminate the need for these variably expressed, cell‐specific promoters, while obtaining specific and robust gene expression in astrocytes. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. We hope to employ these molecules therapeutically where current constitutive gene therapies for Parkinson’s or glioblastoma, for example, could benefit from regulated gene expression.

The brain's astrocytes play key roles in normal and pathological brain processes. Targeting small molecules to astrocytes in the presence of the many other cell types in the brain will provide useful tools for their visualization and manipulation. Herein, we explore the functional consequences of synthetic modifications to a recently described astrocyte marker composed of a bright rhodamine‐based fluorophore and an astrocyte‐targeting moiety. We altered the nature of the targeting moiety to probe the dependence of astrocyte targeting on hydrophobicity, charge, and pKa when exposed to astrocytes and neurons isolated from the mouse cortex. We found that an overall molecular charge of +2 and a targeting moiety with a heterocyclic aromatic amine are important requirements for specific and robust astrocyte labeling. These results provide a basis for engineering astrocyte‐targeted molecular tools with unique properties, including metabolite sensing or optogenetic control.