Microtubules are necessary for proper Reticulon localization during mitosis

Abstract

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

“…The approach presented in this protocol was used to examine the role of microtubules in Endoplasmic Reticulum (ER) reorganization during mitosis in the Drosophila embryo 15 .…”

“…We measured a reduction in ReepB-GFP enrichment to spindles (p<0.001 for all samples at T210) compared to controls (B) (p<0.001 for all samples at T210). This figure has been modified from Diaz et al 15 . Please click here to view a larger version of this figure.…”

“…In the future we would like to produce CNN-RFP 21 , Rtln1-GFP and CNN-RFP; ReepB-GFP transgenic lines to enable automated spindle segmentation. For more information regarding our MATLAB script and analysis pipeline, refer to supplemental materials of Diaz et al 15 .…”

“…This protocol was used to collect, stage, and microinject Drosophila embryos to explore the mechanisms that drive morphological changes in the Endoplasmic Reticulum (ER) during mitosis 15 . Although many studies have outlined the dynamic nature of the ER during the cell cycle 16 , 17 , 18 , 19 , it has been difficult to compare the effects of multiple perturbations across time on ER morphology.…”

“…(B,D) ReepB-GFP in control, and in colchicine. (C,E) Rtnl1-GFP in control, and in colchicine.This figure has been modified from Diaz et al15 . Please click here to view a larger version of this figure.…”