Understanding the role of organelle tethering proteins in ensuring proper cell division

During cell division, a motor protein called dynein anchors to a cortical attachment protein, Num1, before pulling the mitotic spindle into the mother-bud neck. Num1 localization, dependent on the cortical attachment of the endoplasmic reticulum (ER) to the cell membrane (PM), helps guarantee that the mitotic spindle is oriented correctly. This study aims to determine whether or not the tricalbin family of ER-PM attachment proteins, specifically Tcb1 and Tcb2, affect Num1 localization and subsequent mitotic spindle function. Tcb1/2 double mutants were generated through PCR product-mediated homologous recombination. With fluorescent proteins tagged to Num1 and the ER, wide-field fluorescence microscopy was used to observe any resulting changes in ER-PM attachment and Num1 localization. Deletions of Scs2 and Scs22 ER attachment proteins have been found to affect Num1 localization, while the loss of Ist2 has no effect. ER-PM attachment is expected to decrease in tcb1/2∆ mutants. As tricalbins are involved with regulating cortical PI4P levels, which are bound by BAR-domains as in Num1, it is hypothesized that they are involved with the recruitment of Num1. Conversely the deletion of tricalbins may not affect Num1 localization, as preserved Scs2, Scs22 and Ist2 ER-PM attachment proteins could recover the wild-type phenotype. This study aims to understand how ER-PM tethering impacts Num1 localization and subsequent dynein pathway function, along with providing insights into cellular homeostasis and cellular signaling as a whole.