Polarization-Dependent Selective Transport to the Apical Membrane by KIF5B in MDCK Cells

Abstract

Microtubule-based vesicular transport is well documented in epithelial cells, but the specific motors involved and their regulation during polarization are largely unknown. We demonstrate that KIF5B mediates post-Golgi transport of an apical protein in epithelial cells, but only after polarity has developed. Time-lapse imaging of EB1-GFP in polarized MDCK cells showed microtubule plus ends growing toward the apical membrane, implying that plus end-directed N-kinesins might be used to transport apical proteins. Indeed, time-lapse microscopy revealed that expression of a KIF5B dominant negative or microinjection of function-blocking KIF5 antibodies inhibited selectively post-Golgi transport of the apical marker, p75-GFP, after polarization of MDCK cells. Expression of other KIF dominant negatives did not alter p75-GFP trafficking. Immunoprecipitation experiments demonstrated an interaction between KIF5B and p75-GFP in polarized, but not in subconfluent, MDCK cells. Our results demonstrate that apical protein transport depends on selective microtubule motors and that epithelial cells switch kinesins for post-Golgi transport during acquisition of polarity.

LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from theTrans-Golgi Network

“…When examined 10 min after release of the 20°C block, control cells exhibited numerous post-Golgi carriers for p75-GFP, identified as structures devoid of ST-mRFP moving toward the cell periphery with speeds of ϳ0.5 m/s, (Jaulin et al, 2007). By contrast cells treated with LIMK1-KD or cofilin S3A displayed a 90% reduction in the number of such carriers ( Figure 4E and Supplemental Movies 4 and 5, quantification in right bar graph).…”

“…It is very well established that MT motors of the kinesin or dynein families play key roles in the generation and transcytoplasmic transport of tubular and vesicular transporters destined to the apical PM of Madin-Darby canine kidney (MDCK) cells (Kreitzer et al, 2000;Noda et al, 2001;Tai et al, 2001;Allan et al, 2002;Musch, 2004;Jaulin et al, 2007). By contrast, our knowledge of the specific roles of the actin cytoskeleton in intracellular transport routes remains fragmentary, unlike the situation at the PM, where various mechanisms involving the actin cytoskeleton in endocytic routes have been well documented (Erickson et al, 1996;Allan et al, 2002;Luna et al, 2002;Stamnes, 2002;Carreno et al, 2004;Matas et al, 2004;Bonazzi et al, 2005;Perret et al, 2005;Yarar et al, 2005;Egea et al, 2006;McNiven and Thompson, 2006).…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Yet a fourth apical sorting mechanism, also lipid raft-independent, is that of rhodopsin, which binds to the microtubule (MT) motor dynein by cytoplasmic determinants as an apical sorting strategy (Tai et al, 1999). MT motors of the kinesin type are also used by other apical proteins for apical targeting, for example, p75, which requires the plus end kinesin 5B for its apical delivery (Jaulin et al, 2007) and influenza hemagglutinin and Annexin XIIIb, which require the minus end kinesin KIFC3 (Noda et al, 2001). …”

“…This rearrangement of MT is controlled by the polarity protein kinase Par1 (Cohen et al, 2004) (Figures 1 and 3a). Recent work has uncovered the existence of a population of very dynamic MT, emerging from a supranuclear MTOC, in polarized MDCK cells (Jaulin et al, 2007). The repositioning of the MTOC to a supranuclear position is also likely dependent on the activity of Par1 (Cohen et al, 2004) and might be mediated by dynein, as in migrating cells (Etienne-Manneville and Hall, 2003).…”

“…The repositioning of the MTOC to a supranuclear position is also likely dependent on the activity of Par1 (Cohen et al, 2004) and might be mediated by dynein, as in migrating cells (Etienne-Manneville and Hall, 2003). The dual arrangement of MT in polarized MDCK cells accounts for the involvement of both dynein and kinesins in apical transport (Hirokawa, 1998;Tai et al, 1999;Jaulin et al, 2007). Disruption of MT with nocodazole or colchicine disrupts selectively the localization of apical PM proteins (van Zeijl and Matlin, 1990;.…”