Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: Analysis by free-flow electrophoresis

Abstract

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route-specific ligands in combination with temperature shift protocols. Free-flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1-3 min) internalization at 37 degrees C, 125I-ASOR and fluorescein isothiocyanate (FITC)-pIgA were colocalized in common early endosomes. Following a 5-10 min chase of the ligands at 37 degrees C endosomes labeled with 125I-ASOR were separated from endosomes labeled with FITC-pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16 degrees C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I-ASOR or 125I-pIgA were first chased into late compartments at 37 degrees C and the temperature was subsequently lowered to 16 degrees C, biliary secretion of 125I-ASOR-derived counts was arrested, while biliary output of 125I-pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16 degrees C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.

Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature

“…Consequently, transport from late endosomes (pH≤5.6) to lysosomes is prevented at 20°C (Prchla et al 1994). This is in accordance with various other cell types (hepatocytes, BHK, MDCK cells) in which lysosomal degradation does not occur when ligands are internalized at low temperature (Barroso and Sztul 1994;Dunn et al 1980;Ellinger et al 2002). Incubation at low temperature does not affect dextran acidification up to a 15-min chase (Fig.…”

“…Although uptake at 20°C is reduced, HRP is internalized into early endosomes in AtT20 cells (Tooze and Hollinshead 1991), and fluid-phase markers and HRV2 reach late endosomes but not lysosomes in HeLa cells (Kronenberger et al 1997;Prchla et al 1994). In rat hepatocytes, two temperaturesensitive steps along the degradative route have been identified: 1) between early and late endsomes, and 2) between late endosomes and lysosomes (Dunn et al 1980;Ellinger et al 2002;Mueller and Hubbard 1986).…”

Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

“…In HeLa cells, 20°C caused a small reduction of endocytosis, but a near complete block of the removal of internalised dextran-containing late endosome vesicles [ 15 ]. In rat hepatocytes, temperatures of 16–20°C blocked the transition from early to late endosomes, and from late endosomes to lysosomes [ 26 , 27 ]. Uptake and catabolism of endocytic particles continued, but progressively slowed as the temperature decreased from 35 to 20°C.…”

Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

“…Its concomitance with early and late endosomes was verified monitoring the transport of specific ligands of the lysosomal and transcytotic pathways at different temperatures. Separating the organelles involved by FFE, the distinct temperature sensitivity of both the routs could be clearly unveiled .…”