Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

Abstract

Clathrin and clathrin-dependent events are evolutionary conserved although it is believed that there are differences in the requirement for clathrin in yeast and higher vertebrates. Clathrin is a long-lived protein and thus, with clathrin knockdowns only long-term consequences of clathrin depletion can be studied. Here, we characterize the first vertebrate temperature-sensitive clathrin heavy chain mutant as a tool to investigate responses to rapid clathrin inactivation in higher eukaryotes. Although we created this mutant using a clathrin cryo-electron microscopy model and a yeast temperature-sensitive mutant as a guide, the resulting temperature-sensitive clathrin showed an altered phenotype compared to the corresponding yeast temperature-sensitive clathrin. First, it seemed to form stable triskelions at the non-permissive temperature although endocytosis was impaired under these conditions. Secondly, as a likely consequence of the stable triskelions at the non-permissive temperature, clathrin also localized correctly to its target membranes. Thirdly, we did not observe missorting of the lysosomal enzyme beta-glucuronidase which could indicate that the temperature-sensitive clathrin is still operating at the non-permissive temperature at the Golgi or, that, like in yeast, more than one TGN trafficking pathway exists. Fourthly, in contrast to yeast, actin does not appear to actively compensate in general endocytosis. Thus, there seem to be differences between vertebrates and yeast which can be studied in further detail with this newly created tool.

RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells

“…To evaluate the viral entry into HepG2 cells, the silenced cells were infected with a live DNEV 72 hours post transfection to confirm that the target proteins level was reduced as the half-life time of these target proteins is less than 72 hours as shown by previous studies. These proteins possess a half-life of 24–48 hours for GRP78 [66] , [67] , [68] , [69] , [70] , [71] , 18–36 hours for CLTC [72] , [73] , [74] , and 24–34 hours for DNM2 [75] .…”

Diversity of Clathrin Function: New Tricks for an Old Protein

“…The first was the development of temperature-sensitive alleles in yeast, which have been reproduced in worms and mammalian cells (Lemmon et al 1991, Neumann-Staubitz et al 2010, Sato et al 2009, Seeger & Payne 1992. Secondly, mammalian cell lines and Drosophila have been engineered to express modified CLCs that can be crosslinked by cell-permeable bivalent compounds (Foraker et al 2012, Moskowitz et al 2003 or by light exposure (Heerssen et al 2008).…”

SDF-1 Chemokine Signalling Modulates the Apoptotic Responses to Iron Deprivation of Clathrin-Depleted DT40 Cells

“…We designated this cell-line DKO-R ( d ouble k nock o ut cells r esistant to clathrin-depletion) [5] . We have used both of these DKO cell-lines to study clathrin function [5] , [6] , [7] , [8] , [9] .…”