12/25/2023 0 Comments Mical pronunciation![]() Further establishing a link between LITAF and the TRE, epitope-tagged LITAF co-immunoprecipitated with GFP–Pacsin2 ( Fig. 3C) and with GFP–EHD1 ( Fig. 3D). ![]() Indeed, Pacsin2 tubules were rarely seen in untransfected Vero cells but this protein localised to short GFP–LITAF(SLD) tubules in transfected cells ( Fig. S2C), suggesting that the LITAF SLD might be able to induce a tubular recycling compartment. ![]() GFP–LITAF(SLD) partitioned into tubules in Vero, U2OS and COS7 cells ( Fig. S2B). Furthermore, co-expressing HA–ARF6 T44N, which prevents nucleotide exchange ( Macia et al., 2004), enhanced the tubular morphology of GFP–LITAF(SLD)-labelled areas, and also to some extent those of FL GFP–LITAF ( Fig. S2A). LITAF(SLD) also labelled tubules containing HA-tagged ARF6 ( Fig. 3B). Although some GFP–LITAF(SLD) tubules did not label with antibodies for TRE markers, nearly all Pacsin2-positive tubules ( Fig. 3A 90% data from 31 cells in three independent experiments) and MICAL-L1-positive tubules (96% 35 cells three experiments) contained GFP–LITAF(SLD). These tubules resemble the exaggerated tubular recycling endosome (TRE) induced in HeLa cells upon expression of an ARF6 dominant-negative mutant, overexpression of Pacsin2, or either overexpression or depletion of the scission ATPase EHD1 ( Braun et al., 2005 Caplan et al., 2002 Radhakrishna and Donaldson, 1997 Sharma et al., 2009). Partitioning to tubules is a conserved feature of the SLD, since the SLD of cell death involved p53 target 1 (CDIP1), the other LITAF family protein expressed in humans ( Brown et al., 2007 Qin et al., 2016), also localised strongly to tubules ( Fig. S1A,C), whereas FL CDIP1 localised to vesicular compartments ( Fig. S1A,C).Ĭorrelative light electron microscopy (CLEM) confirmed that GFP–LITAF(SLD) associated with linear membrane tubules of uniform diameter (54.2 nm, median 95% confidence limits 52.1–56.0 nm) as well as with endosomes ( Fig. 2). Hence, partitioning into tubules is an inherent behaviour of the SLD, and is modulated within FL LITAF. However, close examination showed that some exogenous ( Fig. S1A,B) and endogenous ( Fig. S1D) LITAF also labelled tubules weakly. In contrast, full-length (FL) LITAF localised strongly to vesicular compartments ( Fig. 1D Fig. S1A,B), previously identified as early and late endosomes ( Ho et al., 2016 Lee et al., 2012 Moriwaki et al., 2001 Qin et al., 2016 Zhu et al., 2013). In HeLaM cells, GFP–LITAF(SLD) or StrepTag–LITAF(SLD) decorated tubules strongly while also labelling the plasma membrane clearly ( Fig. 1D Fig. S1A,B). ![]() Membrane curvature domains often partition to tubular compartments ( Voeltz et al., 2006). Consistent with this, co-depletion of LITAF and CDIP1 impairs integrin recycling and cell migration. Meanwhile, co-depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF and CDIP1 in membrane scission. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC class I and CD59. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. Here, we identify the monotopic membrane protein LPS-induced TNF-activating factor (LITAF) and the related protein cell death involved p53 target 1 (CDIP1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes.
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