LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones.
BACKGROUND
The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones.


RESULTS
Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous.


CONCLUSIONS
The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue.
Jones
Vicky C
VC
Faculty of Life Sciences, The University of Manchester, Manchester, M13 9NT, UK. victoria.c.jones@postgrad.manchester.ac.uk
McKeown
Lynn
L
Verkhratsky
Alexei
A
Jones
Owen T
OT
D000818:Animals; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D014162:Transfection / Q000379:methods; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
C490685:EXOC3 protein, human; D009842:Oligopeptides; D021382:Protein Sorting Signals; D033921:Vesicular Transport Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; D049452:Green Fluorescent Proteins