Biology subjects

Alas, G. C. M.

Publications and source records attributed to Alas, G. C. M..

5 recordsLinked to original sources

bioRxiv2024

Unraveling the Role of cAMP Signaling in Giardia: Insights into PKA-Mediated Regulation of Encystation and Subcellular Interactions

cAMP plays a crucial role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP (EPAC), and cAMP-response element binding protein (CREB) transcription factors, to initiate signal transduction in humans. Even though Giardia has been observed to have only two adenylate cyclases (ACs) and a single cAMP effector, the specific functions of these components are not fully understood. In our previous research, we demonstrated the essential role of AC2-dependent cAMP signaling in the upregulation of the encystation program. Using NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of PKAr and PKAc. In this study, our objectives are twofold: first, we used newly developed Split-Halo to examine subcellular interactions of GlPKAr and GlPKAc; second, we investigated whether PKAc regulates encystation markers. Our findings revealed distinct subcellular locations where GlPKAr and GlPKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytosol. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Additionally, interaction occurred at an unidentified cell compartment between twelve to sixteen hours of encystation and persisted to the cyst stage. Knockdown of GlPKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in transcriptional regulation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its pivotal role in governing Giardias encystation process.

developmental biology↗

bioRxiv2023

Encystation stimuli sensing mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2- dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite.

developmental biology↗

bioRxiv2021

An early signaling transcription factor regulates differentiation in Giardia

Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia cells shift from G1+S phase of the cell cycle to G2+M. By 2-4 hours, cyst wall proteins are upregulated, indicating that key regulatory steps occur within the first four hours of encystation. However, all characterized transcription factors (TFs) in Giardia have only been investigated at later time points of encystation. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early upregulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, GLP4 that increases rapidly after 30 min of encystation stimuli and downregulates encystation specific markers, including Cyst Wall Proteins (CWPs) and enzymes in the cyst N-acetyl galactosamine (GalNAc) pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1 resulting from the activation of MYB2, a TF previously linked to encystation in Giardia. GLP4 upregulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 upregulates MYB2 and promotes encystation while overexpression of GLP4 downregulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell cycle regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway. ImportanceTransition between life cycle stages is a common feature among parasitic pathogens and its regulation must be optimized to balance persistence of infection with transmission. The early transcription factors (TFs) regulating commitment to differentiate are totally unknown in Giardia. In this work, we identified GLP4, a previously uncharacterized GARP-like TF, as an early-acting transcriptional repressor that inhibits G1+S cells from entering the encystation pathway. GLP4 is therefore a key regulator controlling the balance between proliferative growth and terminal differentiation into infective cysts.

developmental biology↗

bioRxiv2021

The Giardia lamellipodium-like ventrolateral flange supports attachment and rapid cytokinesis

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The microtubule cytoskeleton plays a well characterized role in attachment via the ventral adhesive disc, whereas the role of the unconventional actin cytoskeleton is controversial. We identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin and is enriched in the ventrolateral flange (VLF), a lamellipodium-like membrane protrusion at the interface between parasites and attached surfaces. Live imaging revealed that the VLF grows to ~1 m in width after cytokinesis, then remains size-uniform in interphase, grows during mitosis, and is resorbed during cytokinesis. A Flangin truncation mutant stabilizes the VLF and blocks cytokinesis, indicating that the VLF is a membrane reservoir supporting rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions, GlRac, the sole Rho family GTPase in Giardia, was localized to the VLF. Knockdown of Flangin, actin, and GlRac result in VLF formation defects indicating a conserved role for GlRac and actin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites challenged with fluid shear force in flow chambers had a reduced ability to remain attached, indicating a role for the VLF in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that is particularly important during mitosis when the parental ventral disc begins disassembly in preparation for cytokinesis. ImportanceThe ventrolateral flange (VLF) is a lamellipodium-like structure found at the host-parasite interface that has long been thought to be involved in parasite attachment. The proteins responsible for building the VLF have remained unidentified precluding manipulation of the VLF to determine its role in Giardia biology. We identified Flangin, a novel actin associated protein that localizes to the VLF, implicating Giardia actin in VLF formation. We demonstrate that: 1.) Flangin, actin, and GlRac are required for VLF formation, 2.) the VLF serves as a membrane reservoir to support Giardias incredibly fast cytokinesis, and 3) the VLF augments attachment, which is critical to parasitism. The microtubule-based adhesive ventral disc and the actin-based ventrolateral flange represent redundant means of maintaining attachment, the presence of redundant systems illustrate the importance of attachment to the lifestyle of this ubiquitous parasite.

cell biology↗

bioRxiv2017

14-3-3 Negatively Regulates Actin Filament Formation In The Deep Branching Eukaryote Giardia lamblia

The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin, this function has been previously attributed to sequestration of phosphorylated cofilin. The deep branching eukaryote Giardia lamblia lacks cofilin and all other canonical actin-binding proteins (ABPs), and 14-3-3 was identified as an actin-associated protein in Giardia, yet its role in actin regulation was unknown. Gl14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia's sole Rho family GTPase, GlRac, modulates Gl14-3-3's association with actin, providing the first connection between GlRac and the actin cytoskeleton in Giardia. Giardia actin contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a negative regulator of actin filament formation.\n\nImportanceGiardia lacks canonical actin binding proteins. 14-3-3 was identified as an actin interactor but the significance of this interaction was unknown. Loss of 14-3-3 results in ectopic short actin filaments, indicating that 14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that 14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardias sole Rho family GTPase, GlRac, this result provides the first mechanistic connection between GlRac and actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between 14-3-3 and actin suggesting that 14-3-3 is regulating multiple actin complexes. Overall, we find that 14-3-3 is a negative regulator of actin filament formation in Giardia.

cell biology↗