NOVEL NUCLEOLAR LOCALIZATION OF SYNAPTOPODIN-2 ISOFORM IN PROLIFERATING HT-29 CELLS

Abstract

Members of the Synaptopodin family regulate [alpha]-actinin and actin-bundling activity, in an isoform specific manner (Asunama et al., 2005). The ability to affect actin polymerization and bundling highlights the critical role that synaptopodin members play in normal and pathologic cells. Synaptopodin-2 (Synpo2) is one member of the synaptopodin family that is capable of inducing actin polymerization (Chalovich [and] Schroeter, 2010), suggesting an important role in cell migration, adhesion, division, and development. Synpo2 has also been described as a valuable biomarker for several invasive cancers due to its ability to alter cell motility in response to external stimuli (Kai et al, 2015). Although most data support the Synpo2's role as a tumor suppressor (Xia et al., 2018; Liu, et al., 2018; Kai et al, 2012; Cebrian et al., 2008; Sanchez-Carbayo et al., 2003; Jing et al., 2004; Lin et al., 2001), it has been reported that under certain conditions, the loss of Synpo2 expression led to decreased motility and invasiveness (De Ganck et al, 2009). These contradictory reports of the influence of the expression of Synpo2 in cell migration and tumor stimulation/suppression demonstrate the need for further research. Synpo2 exists as five isoforms, which are the result of alternative splicing and cause differences at the N-terminal end of the proteins (Chalovich [and] Schroeter, 2010). Each isoform has a unique response to different chemokinetic stimuli (Kai et al., 2012). To improve the understanding of individual roles that these isoforms play in human colorectal cancer cells, this study considers the difference in expression of specific Synpo2 isoforms in HT29 cells. We observed intense and irregular staining in areas within the nucleus, with a pattern that was 90% correlated with markers of nucleoli. Furthermore, transcriptional inhibition resulted in a loss of nuclear stain. Induction of differentiation in HT29 cells with sodium butyrate, resulted in a loss of nuclear expression detected by the synaptopodin 2 antibody, but showed an increase in previously undetected cytoplasmic expression. RT-PCR and Western blot analysis showed that HT29 cells lacked Synpo2 isoform B, but expressed its smaller truncated isoform, myopodin. Nuclear fractions detected the expression of a smaller cross-reactive protein at approximately 55 kDa. This unexpected protein recognized by our affinity purified antibody could potentially be a previously uncharacterized Synpo2 isoform. The nucleolar expression detected though immunofluorescence suggests a highly critical role in normal cell function.