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Trop-2-dependent Recruitment of PKCα to the Cell Membrane

Marco Trerotola and Saverio Alberti

The TROP2 gene (TACSTD2) (1,2) encodes a transmembrane calcium signal transducer (3), that is characterized by a non-canonical EGF-like repeat and by a thyroglobulin domain (1,4-6). The Trop-2 EGF-like and thyroglobulin domains demonstrate significant similarity to the corresponding regions of Trop-1/Ep-CAM, nidogen and IGF-binding proteins (1,4,5). The TROP2 gene is highly conserved across species (1,4), consistently with a strong evolutionary pressure for a conserved functional role.

PKCα is activated in a Trop-2-dependent manner. As activated PKCα translocates to the cell membrane (7), a Trop-2-dependent recruitment of PKCα to the cell membrane was visualized by confocal time-lapse microscopy analysis of living MTE 4-14 cells transfected with Trop-2-mRFP1 and with PKCα–EGFP. Trop-2-mRFP1 mainly localized at the cell membrane rim, in the endoplasmic reticulum and in vesicular formations, as in natively expressing cancer cells. Dynamic colocalization of PKCα with Trop-2 in membrane ruffles and podosomes was shown (Movie 1). Recursive waves of strikingly coordinated relocation of PKCα and Trop-2 to specific domains of the cell membrane were observed every few to several minutes, morphological analysis suggesting a tight relationship with podosome dynamics (Movie 1). No such changes were detected in control cells transfected with PKCδ–EGFP (Movie 2).

Material and methods


The murine immortalized MTE 4-14 cell line (8) was maintained in DMEM supplemented with 10% fetal calf serum.


The pEYFP expression vector was obtained from Clontech (Palo Alto, CA). The pGFP3-PKC-α-WT and pGFP3-PKC-δ-WT expression vectors were generously provided by Jae-Won Soh (9). The expression vector pmRFP1 (10) was kindly provided by R. Tsien. Chimeric proteins between Trop-2 and mRFP1 were constructed as described (11,12), after substituting the mRFP1 (XhoI / KpnI) coding region for that of EYFP in pEYFP.

Confocal time-lapse microscopy

Cells cultured on glass slides were incubated in Leibovitz’s F15 culture medium devoid of phenol red and bicarbonate, supplemented with 10% FBS, 50 UI/ml Penicillin/Streptomycin and 2 mM N-acetyl-cysteine (Sigma) to reduce free-radical damage. Cells were viewed with an LSM-510 META (Zeiss) confocal microscope. Images were captured at 1 min intervals. Excitation of EGFP was at 488 nm; mRFP1 was excited at 546 nm.

Movie legends

Movie 1 – MTE 4-14 cells transfected with Trop-2-mRFP1 and with PKCα–EGFP. Cytoplasmic vescicles containing Trop-2-mRFP1 are useful landmarks for the localization of PKCα–EGFP (empty cytoplasmic regions). Trop-2-mRFP1 and PKCα–EGFP dynamically colocalize in membrane ruffles and podosomes.

Movie 2 – MTE 4-14 cells transfected with Trop-2-mRFP1 and with PKCδ–EGFP. Cytoplasmic vescicles containing Trop-2-mRFP1 are useful landmarks for the localization of PKCδ–EGFP (empty cytoplasmic regions). No significant colocalization of Trop-2-mRFP1 and of PKCδ–EGFP was observed.


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