The Endoplasmic Reticulum

by John Runions

School of Life Sciences, Oxford Brookes University,Oxford, UK. OX3 0BP

The Endoplasmic reticulum in a hypocotyl cell of Arabidopsis. Some regions of ER overlay thick actin cytoskeleton bundles and they remodel very quickly - these are seen in this case as fast moving diagonal stripes generally moving from top left to bottom right.
Other regions persist in a reticulate pattern for extended periods of time. The ER is marked with GFP-HDEL and the timelapse is 15 minutes.
In plant cells, the endoplasmic reticulum (ER) occurs as a system of membrane tubules and sheets (cisternae) that is distributed throughout the cytoplasm.  It forms a network that is continuous with the nuclear envelope [1].  The ER is generally flattened in a 2-dimensional plane and in most cell types occupies a peripheral position (cortical) just inside the plasma membrane (as, indeed, does the cytoplasm that contains it).  In 3-dimensional reconstructions, it appears as a net-like basket that completely encloses the cellular space.  Large vacuolar spaces are traversed by cytoplasmic strands (transvacuolar strands) that also contain ER.  Examined at higher magnification, the ER is seen to be composed of two thin, parallel unit membranes with lipid bilayer structure that enclose a space - the ER lumen.
Ultrastructural studies conducted by transmission electron microscopy reveal that the ER is composed of two types.  Cisternal ER that is covered in ribosomes is termed rough endoplasmic reticulum (RER) and the tubular form that contains fewer ribosomes is known as smooth endoplasmic reticulum (SER).  Both types of ER occur in all cells however RER occurs relatively more in cells that are making and storing proteins while SER occurs more so in cells that are actively producing and secreting lipids and synthesizing membranes. 
ER is a major component of the cell’s secretory pathway which is a more or less continuous membrane system of organelles and vesicles that spans the space from the sites of membrane synthesis to their final functional location.  Membrane-associated proteins and those destined for locations such as the storage vacuole are produced by translation of the mRNA signal at ribosomes and inserted directly into the ER via the translocon pore (co-translational insertion).  Proteins to be transported in this manner bear an amino terminal  signal/transit peptide sequence that directs them into the ER before it is cleaved from the proprotein within the lumen.  Simple modifications such as protein glycosylation may occur within the ER but most proteins continue transiting the secretory pathway where they are further modified at the next station - the Golgi body.  The ER also sequesters calcium ions (Ca2+) and therefore plays a role in signalling and developmental processes that utilize calcium.
Fluorescent protein aided marking of the ER
From the mid-1990s, we have witnessed the rise of fluorescent proteins (FPs) in cell biological studies.  The reason for this renaissance is quite simply that green fluorescent protein (GFP) and its alternatively-coloured relatives are genetically encoded within cells and therefore allow observation of organelles within living cells.  For plant biologists the paradigm shifted during the period from 1995-97 when various groups including Petra Boevink and her colleagues and Jim Haseloff and his colleagues first were successful in getting GFP expression in plant cells and then of targeting that expression to the ER [2,3,4,5,6,7].  The strategy for marking a specific organelle is to modify the DNA of the FP gene so that it contains a targeting sequence.  In the case of ER, this required not only addition of the 5’-signal for directing the nascent protein through the translocon into the ER but, as well, addition of a 3’-KDEL or HDEL signal sequence which causes the GFP to be retained within the lumen of the ER.

Availability of this type of genetic construct opened the door for secretory pathway research because the ER became highly fluorescent.  The first remarkable finding was of just how mobile the whole endomembrane system (ER, Golgi bodies, vesicles etc.) is.  The ER constantly remodels with some strands being in fast motion and other reticulate and tubular areas moving over a slower relatively longer time course.  In older studies based on examination of ER structure in fixed and stained cells, the motility and constant re-modelling of the whole system was not observed or even predicted.  Why and how does the ER move and re-model the way that it does?  This is the question that is always asked when for the first time a student sees the way that it moves around in living cells observed with the confocal microscope.  The most parsimonious explanation for constant movement is that it is driven by energy input from the cell as a means of distributing proteins from their site of synthesis to their site of action within the cell.  This is quite likely as some plant cells are quite large.  Movement of the ER and other secretory pathway organelles like Golgi bodies is actin cytoskeleton dependant [8].  A recent study [9] employed photoactivatable GFP to study the movement kinetics of the ER membrane and its association with the motile Golgi bodies.  The finding was that the ER membrane protein calnexin diffuses freely even when rapid movement of the ER and Golgi bodies has been abolished by treatment with LatrunculinB to depolymerize the actin cytoskeleton.

The second advantage of using FPs is, of course that they can be fused to any protein of interest and used to observe its targeting within a cell.  This technique has been employed for many different ER marking proteins (see Table 1).  Some of the proteins fused to fluorescent proteins in these types of study have known or predicted function and others are proteins with unknown function that mark the ER and which were discovered in large-scale screens [10,11]. The proteinlocalisation data base (ProtLocDB) hosted bythe Scottish Crop Research Institute lists 140 plant proteins that markthe ER in random fluorescent protein fusions. Nelson and co-workers[12] have recently published a set of 11 different ER-localizedproteins that they will supply in various different colours (cyan,green, yellow, cherry). This same paper details markers in all coloursfor most organelles.
Fluorescent Protein Probes for the Endoplasmic reticulum


Table FP probes for the ER

References

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