Phillips, B. A. | bioRxiv Science

bioRxiv2022

Katnip is required to maintain microtubule function and lysosomal delivery to autophagosomes and phagosomes

The efficient delivery of lysosomes is essential for many cell functions, such as the degradation of unwanted intracellular components by autophagy and the killing and digestion of extracellular microbes within phagosomes. Using the amoeba Dictyostelium discoideum we find that cells lacking Katnip (Katanin interacting protein) have a general defect in lysosome delivery and whilst able to make autophagosomes and phagosomes correctly are then unable to digest them. Katnip is largely unstudied yet highly conserved across evolution. Previously studies found Katnip mutations in animals cause defects in cilia structure. Here we show that Katnip plays a general role in maintaining microtubule function. We find that loss of Katnip has no overall effect on microtubule dynamics or organisation, but is important for the transport and degradation of endocytic cargos. Strikingly, Katnip mutants become highly sensitive to GFP-tubulin expression, which leads to microtubule tangles, defective anaphase extension and slow growth. Our findings establish a conserved role for Katnip in the function of all microtubules, not just cilia as previously reported. We speculate this is via a key function in microtubule repair, required to maintain endosomal trafficking and lysosomal degradation.

cell biology↗

bioRxiv2019

Co-ordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialised cup shaped protrusions. To effectively capture and internalise their targets, these cups are organised into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac and phospholipid interactions to localise at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, RGBARG shapes the protrusion by driving Rac activation at the rim whilst suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of co-ordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

cell biology↗