J. a significant hereditary renal disease in childhood, with estimated prevalence of 1 1 in 20 000 live births. A carrier frequency of up to 1:70 has been proposed (1). Classic ARPKD is generally defined as an infantile disorder with diagnosis or within the neonatal period, which presents with greatly enlarged echogenic kidneys. Some patients are diagnosed after neonatal age, which form an older group. In the classic group, most patients have renal insufficiency and ESRD at birth. The older group more often involves liver manifestations, including hepatosplenomegaly, variceal bleeding and cholangitis. Some patients with the diagnosis of ARPKD at 1 year of age with nephromegaly exhibit slow renal function decline over 20 years with only minimally enlarged kidneys at ESRD and markedly atrophic kidneys following renal transplantation (2). The slow progression of renal disease is likely due to increasing fibrosis, rather than the development of cysts. Genetic linkage studies indicate that mutations within a single gene, by a germ line mutation in the PCK rat results in abnormalities in primary cilia of cholangiocytes (11,12). Some reports have also shown FPC localization on the basal body (7,9) and plasma membrane (10,13). The large extracellular domain of FPC is presumed to bind to a yet unknown ligand(s). The C-terminal ICD of FPC harbors a nuclear localization signal and may be cleaved and translocated to the nucleus in cells stimulated with protein kinase C activator or Ca2+ ionophore which increases intracellular Ca2+ (14). FPC interacts with polycystin-2 (PC2), whose gene is mutated in the ADPKD. This interaction may RIPGBM occur between RIPGBM the ICD of FPC and the amino terminal ICD of PC2 (15) or through a third binding partner, such as kinesin-2 (16). Downregulation of PC2 in the mice with hypomorphic alleles (mice (15), all of which support the hypothesis that there is a common mechanism underlying cystogenesis in AD- and ARPKD. In ciliated terminally differentiated cells, FPC may form a complex with polycystins and modulate Ca2+ signaling in response to extracellular fluid flow stimulation (13). Misorientation of the mitotic spindle has recently been proposed to cause cyst formation in rats, an orthologous model of human ARPKD (17,18), although the importance of spindle orientation in cystogenesis was questioned because of the absence of cysts in knockout mice with misorientated mitotic spindles at 1 year of age (19). The role of FPC during cell division and differentiation remains unclear. In the present study, we found that RIPGBM endogenous FPC localizes to the centrosome and mitotic spindle in human and mouse renal epithelial cells by using two specific FPC antibodies. Knockdown of FPC in canine and mouse epithelial cells by short-hairpin-mediated RNA (shRNA) leads to multipolar spindles in mitotic cells and multiple centrosomes in interphase cells. Multiple centrosomes can also be detected in tubular epithelial cells in ARPKD kidneys, similar to those in human ADPKD kidneys (20). Thus, we report a new subcellular location for FPC and a novel role of FPC in the regulation of centrosome duplication and mitotic spindle assembly. RESULTS Construction of a full-length human FPC expression construct The longest ORF of is transcribed from 67 exons that encode a transmembrane protein containing 4074 amino acids. Thus, we strategically amplified four overlapping DNA fragments by PCR from cDNA synthesized from human fetal kidney RNA using specific FPC primers. These four fragments were then assembled sequentially utilizing unique restriction enzyme sites shared by the overlapping fragments in pcDNA4/TO/myc-His B vector to make a full-length cDNA expression construct with a myc/His tag fused in frame at the C-terminus of FPC (Fig.?1A). F4 set of primers amplified two different sizes of PCR products and the expected longer product was used for making the full-length construct. Open Col1a1 in a separate window Figure?1. Generation and characterization of inducible full-length FPC stable expressing cell lines in T-REx 293 (hFPCT) and LLC-PK1 (hFPCL) (ACC) and a new FPC C-terminal antibody 4883 (CCG). (A) Scheme of cloning strategy for the longest human cDNA. Four overlapping fragments encoding the entire open-reading frame of were individually amplified and sequentially ligated into pcDNA4/TO/myc-His B vector. (B and C) Characterization of a tetracycline (Tet) inducible cell line stably expressing full-length FPC in T-REx 293 cells (hFPCT) (B) and LLC-PK1 (hFPCL) cells (C) by western blotting. After induction, a high molecular weight band (arrow) can be.