Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Abstract

It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling

“…More recently, actin filaments were proposed to facilitate endocytosis of water channels but not to be required for exocytosis in the toad bladder (28). In some cells, disruption of the actin cytoskeleton inhibits apical endocytosis without affecting basolateral endocytosis (29,30). Another study showed variable effects on receptor-mediated endocytosis after either depolymerization or polymerization of actin filaments in different cell types (31).…”

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities*

“…Although the concentration of monomeric actin in cells is in excess of the critical concentrations for assembly at both barbed and pointed ends, actin polymerization occurs predominantly at fast growing barbed ends (1,2). Indeed, the actin barbed end-binding drug cytochalasin D inhibits many cellular phenomena, including cell migration, cell adhesion, and endocytosis (3)(4)(5). Six actin-nucleating proteins, including Arp2/3, formins, spire, cordon-bleu, leiomodin 2 (Lmod2), and JMY, have been described and play important roles in enhancing actin polymerization from barbed ends of filaments in vivo (6 -11).…”

“…Spire, cordon-bleu, and JMY contain tandem G-actin-binding WH2 motifs, thereby tethering multiple actin molecules into an oligomer that acts as a nucleus (8 -10). Leiomodin2 (Lmod2), a member of the tropomodulin (Tmod) 3 actin filament pointed end capping protein family, shares the domain organization of Tmods but contains a C-terminal extension of ϳ150 amino acids that includes an actin-binding WH2 domain (11,14). Lmod2 is a potent nucleator of actin polymerization, and both the Tmod-related and WH2 domains are necessary for this activity (11).…”

Progress in Cell Cycle Research

“…Strong evidence for the involvement of the actin cytoskeleton in the endocytic pathway comes from genetic studies on Dictyostelium discoideum, where myosin I proteins have been shown to be involved in endocytosis, pinocytosis, and phagocytosis (175)(176)(177). Depolymerisation of actin filaments also inhibited the early stages of endocytosis from the apical surface of polarised epithelial cells whilst having no effect on the basolateral surfaces (178,179). It has been suggested that a myosin motor aids the transport of membrane components along the microvillus and into coated pits for internalisation (178,180) and might also playa role in pinching off clathrin coated vesicles (178).…”

“…Other arguments however are more pertinent and stringent, such as the observation that micro injection of active CK2 into Xenopus oocytes accelerates meiotic maturation induced by mitosis-promoting factor [178], and the inhibition of serum-induced cell cycle progression by microinjection of antibodies against the ~-subunit of CK2 into Go-synchronised human fibroblasts [179]. Incidentally this latter result supports the view that the competent form of CK2 in this case is the canonical tetramer including the ~-subunit, and not the free catalytic subunits.…”