Ingham, P. | bioRxiv Science

bioRxiv2024

The Basal Ciliary but not Cytosol PKA Specifically Regulates HH Pathway Downstream of Smoothened

Effectors of the vertebrate Hedgehog (HH) signaling pathway are organized through primary cilia (PC) that grow and retract in lockstep with the cell cycle in response to extracellular signals. Protein kinase A (PKA), a kinase with ubiquitous distribution in most cells, functions as a specific negative regulator of the HH pathway. Its functional specificity in the HH pathway has been suggested to be controlled by cAMP in the PC. However, the regulation of PKA and its functions in PC remain unclear, in part due to the lack of observation of PKA localization in PC during HH resting state as well as conflicting reports of the dynamic changes of cAMP in cilia and HH pathway activity. To address this issue, we have developed a ciliary-localized FRET-based A-kinase activity probe (Nphp3N-AKAR2-CR) as an improved biosensor for monitoring real-time PKA activity in the PC of both cultured cells and living zebrafish embryos. Although the PKA catalytic subunit (PKA-C) was not observed in PC, basal PKA activity in cells could be detected with this probe. In addition, we have found that only ciliary-targeted PKA and not cytosolic PKA, can modulate the HH pathway, even when the integrity of the PC is disrupted. Notably, ciliary PKA activity was barely changed either by inhibition or activation of the HH pathway at the level of Smoothened (SMO), the obligate HH signal transducer. Moreover, we found that even low concentration of the adenylyl cyclase agonist forskolin (FSK) can efficiently inhibit the HH pathway in the presence of the constitutively active variant SMOA1, suggesting that the activation of the HH pathway by SMO may not be due solely to direct regulation of PKA activity in the PC.

cell biology↗

bioRxiv2017

Dissemination of Cryptococcus neoformans via localised proliferation and blockage of blood vessels

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life-threatening cryptoccocal meningitis, predominantly within immunocompromised individuals. Cortical infarcts are observed in as many as 30% of cryptococcal meningitis cases, being particularly common in severe infection. Limited clinical case studies suggest infarcts are secondary to vasculitis and blood vessel damage caused by cryptococcal infection. However, the cause of infarcts in cryptococcal infection has not been determined. To examine potential causes of vascular damage and cryptococcal dissemination in cryptococcal infection, the zebrafish C. neoformans infection model was used. We demonstrate that spread of cryptococci from the vasculature occurs at sites where cryptococci grow within the blood vessels, originating from a single or small number of cryptococci. We find that cryptococcal cells become trapped within the vasculature and can proliferate there resulting in vasodilation. Localised cryptococcal growth in the vasculature is also associated with sites of dissemination - in some cases simultaneously with a loss of blood vessel integrity. Using a cell-cell junction protein reporter (VE-cadherin) we identified sites dissemination associated with both intact blood vessels and where vessel rupture occurred. Thus, we have identified a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of the vascular damage and cortical infarction observed in cryptococcal meningitis.\n\nAuthor summaryHuman infection by the fungal pathogen, Cryptococcus neoformans, can lead to life-threatening cryptococcal meningitis. In severe cases of cryptococcal meningitis, a lack of blood supply can cause tissue death and a resulting area of dead tissue (infarct) in the brain. Although vasculature inflammation in known to occur in cryptococcal meningitis, the cause of infarcts in unknown. Using a zebrafish model of cryptococcal infection, the growth and dissemination of fungal cells was observed over time. We show that cryptococcal cells become trapped and proliferate in the vasculature, resulting in cryptococcoma that damage the blood vessels. We propose that vessel damage results from increased blood pressure caused by cryptococci blocking blood vessels suggesting that the vascular damage that ensues on cryptococcoma formation may in turn be a cause of infarct formation seen in cryptococcal meningitis.

microbiology↗