Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement

Inflammation is critical for infection control and acts as an arsenal defense mechanism against invading microbes through activation of the host immune system. It works via its inflammasome components to sense the dangerous invading microorganism and send messages to the immune system to destroy them. To date, the function of bovine macrophage inflammasome and its relationship with actin has not been identified. This study aimed to investigate the activation of bovine inflammasome by phase one flagellin from Salmonella typhimurium and its interaction with actin. Bovine monocyte-derived macrophages were prepared and challenged with S. typhimurium SL1344 phase one flagellin. The results demonstrated the relationship between the flagellin-based activation of inflammasome and actin rearrangement. The flagellin-based activation of inflammasome promoted the activation and co-localization of F-actin and the inflammasome complex. Actin was remodeled to different degrees according to the stage of inflammasome activation. The actin redistribution varied from polymerization to filopodia, while at the stage of pyroptotic cell death, actin was broken down and interacted with activated inflammasome complexes. In conclusion, flagellin-dependent inflammasome activation and actin localization to the inflammasome at the stage of pyroptotic cell death may be of importance for appropriate immune responses, pending further studies to explore the exact cross-linking between the inflammasome complex and actin.

TIAM-1/GEF can shape somatosensory dendrites independently of its GEF activity by regulating F-actin localization

Dendritic arbors are crucial for nervous system assembly, but the intracellular mechanisms that govern their assembly remain incompletely understood. Here, we show that the dendrites of PVD neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine-rich transmembrane (LRR-TM) receptor. DMA-1/LRR-TM interacts through a PDZ ligand motif with the guanine nucleotide exchange factor TIAM-1/GEF in a complex with act-4/Actin to pattern higher order 4° dendrite branches by localizing F-actin to the distal ends of developing dendrites. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. A partially redundant pathway, dependent on HPO-30/Claudin, regulates formation of 2° and 3° branches, possibly by regulating membrane localization and trafficking of DMA-1/LRR-TM. Collectively, our experiments suggest that HPO-30/Claudin localizes the DMA-1/LRR-TM receptor on PVD dendrites, which in turn can control dendrite patterning by directly modulating F-actin dynamics through TIAM-1/GEF.

UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

Many axon guidance ligands and their receptors have been identified, but it is still unclear how these ligand-receptor interactions regulate events in the growth cone, such as protrusion and cytoskeletal arrangement, during directed outgrowth in vivo. In this work, we dissect the multiple and complex effects of UNC-6/Netrin on the growth cone. Previous studies showed that in C. elegans, the UNC-6/Netrin receptor UNC-5 regulates growth cone polarity, as evidenced by loss of asymmetric dorsal F-actin localization and protrusion in unc-5 mutants. UNC-5 and another UNC-6/Netrin receptor UNC-40/DCC also regulate the extent of protrusion, with UNC-40/DCC driving protrusion and UNC-5 inhibiting protrusion. In this work we analyze the roles of UNC-6/Netrin, UNC-40/DCC, and UNC-5 in coordinating growth cone F-actin localization, microtubule organization, and protrusion that results in directed outgrowth away from UNC-6/Netrin. We find that a previously-described pathway involving the UNC-73/Trio Rac GEF and UNC-33/CRMP that acts downstream of UNC-5, regulates growth cone dorsal asymmetric F-actin accumulation and protrusion. unc-5 and unc-33 mutants also display excess EBP-2::GFP puncta, suggesting that MT + end accumulation is important in growth cone polarity and/or protrusion. unc-73 Rac GEF mutants did not display excess EBP-2::GFP puncta despite larger and more protrusive growth cones, indicating a MT-independent mechanism to polarize the growth cone and to inhibit protrusion, possibly via actin. Finally, we show that UNC-6/Netrin and UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of F-actin asymmetry and MT + end entry. Our data suggest a model in which UNC-6/Netrin polarizes the growth cone via UNC-5, and then regulates a balance of pro- and anti-protrusive forces driven by UNC-40 and UNC-5, respectively, that result in directed protrusion and outgrowth.

Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells

Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin− SCA-1+ KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.