1,721,041 research outputs found
Gene stacking and stoichiometric expression of ER-targeted constructs using "2A" self-cleaving peptides
No full textSimultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or "gene stacking") have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments
Observing ER Dynamics over Long Timescales Using Light Sheet Fluorescence Microscopy
No full textThe recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock
Quantitation of ER morphology and dynamics
Full text linkThe plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available
Studying Plant ER-PM Contact Site Localized Proteins Using Microscopy
No full textAs in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for studying EPCS structures and identifying possible EPCS-associated proteins. These include using artificially constructed reporters, GFP tagged protein expression followed by image analysis, and immunogold labelling at the ultrastructural level. In combination, these methods can be used to identify the location of putative EPCS proteins, which can aid in predicting their potential subcellular function
Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells
Full text linkOptical tweezers have been used to trap and micromanipulate several biological specimens ranging from DNA, macromolecules, organelles to single celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence (TIRF) microscopy can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells
Characterization of Proteins Localized to Plant ER-PM Contact Sites
No full textLike in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for identifying possible EPCS-associated proteins. These include GFP-tagged protein expression followed by image analysis, and immuno-gold labeling at the ultrastructural level. In combination, these methods can be used to identify the localization of putative EPCS proteins as well as used to postulate their subcellular function
Quantitation of ER structure and function
No full textThe plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to a thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available
Modifying N-linked glycosylation in tobacco to produce human-type post-translational modifications
Full text linkPlant cells are a capable system for producing high value proteins for a variety of applications, and challenge popular host organisms such as mammalian cells, bacteria or yeasts. However, plants do not perform post translation protein modification in the same manner as mammalian cells, most critically in terms of N-linked glycosylation. This can impact both protein functionality and stability, key for human therapeutics intended for production in plants. This obstacle can be approached by creating a plant-based system capable of “humanizing” proteins of interest in terms of N-linked glycosylation, resulting in plant-produced proteins with a glycan profile as would occur in a mammalian host system, making them more suitable for therapeutic applications.
For this, four human glycosylation enzymes (HuGEs) involved in N-linked glycosylation N- acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β-1,4-galactosyltransferase (B4GALT1), and α-2,6-sialyltransferase (ST6GAL) need to be expressed in plant cells. As glycosylation is a stepwise process, enzymes need to localise to either medial or trans-Golgi body cisternae. For this, a protein targeting strategy of replacing the mammalian N-terminal Golgi targeting domains on HuGEs (Cytoplasmic-Transmembrane-Stem (CTS) regions) with plant–specific domains, MUR3 or FUT13, were tested transiently and were successful in redirecting localisation. This was conducted via high-resolution dynamic confocal microscopy using an analysis pipeline based upon distance between peak fluorescence intensity, relative to known Golgi markers MNS1 and ST. This analysis demonstrated that the modified MUR3-GNTIV and MUR3-GNTV were successfully targeted to the medial-Golgi cisternae while FUT13-ST6GAL and FUT13-B4GALT1 were targeted to trans-Golgi cisternae.
Following successful expression and targeting of the four HuGEs transiently, these four enzymes were combined into two expression cassettes for stable plant transformation. These consisted of a N-terminal CTS domain, the catalytic subunit of the first enzyme, a linker domain, and the second enzyme catalytic subunit without attached fluorescent protein. This halved the insert number necessary to generate stable glycomodified lines, transformed using the modified HuGEs in wild type and plant-glycan deficient (ΔXFT) lines of N. benthamiana. Following growth, resulting seedlings of this line were successfully screened for presence of each of the four HuGE catalytic domains by RT-PCR against a 300BP sequence specific to each enzyme. Human proteins of interest LAL, IgE and IgG were then transiently expressed in these glycomodified lines and lectin binding assays performed against the plant extracts, either dot blot or by SDS-PAGE. The results of the lectin binding assays potentially indicate altered glycomodification in the HuGE stable lines but are ultimately inconclusive
The role of Atgolgin-84A at the plant ER-Golgi interface
Full text linkThe eukaryotic cell is compartmentalised into an endomembrane
system organised into several organelles. Some of these organelles, namely
endoplasmic reticulum (ER) and Golgi bodies, are part of a so-called
secretory pathway where vital cell components travel to reach the correct
final destination or are recycled for further processing. In the plant cell
cytoplasm there are numerous stacks of membrane bounded cisternae, each
of which constitutes a discrete Golgi body. Golgi bodies are responsible in
part for the processing of proteins received from the ER and their distribution
to the plasma membrane and other compartments. Exactly how this motile
structure is maintained while providing vital functions for the cell is still poorly
understood. The ER is physically connected to Golgi bodies, and Golgi matrix
components, such as golgins, have been identified and suggested to function
as putative tethering factors. Golgins are proteins anchored to the Golgi
membrane by the C-terminus either through transmembrane domains
(TMDs) or interaction with small regulatory GTPases. The golgin N-terminus
contains long coiled-coil domains which consist of a number of α-helices
wrapped around each other to form a structure similar to a rope being made
from several strands, reaching into the cytoplasm.
Atgolgin84A may act as tethering factor at the ER-Golgi interface and
within the Golgi stack. In animal cells golgins are also implicated in specific
recognition of cargo at the Golgi. In plants, there is no clear evidence for the
localisation of Atgolgin-84A at the Golgi. To investigate Atgolgin-84A
subcellular localisation and putative function as a tether, fluorescent fusions
to Atgolgin-84A and Atgolgin-84A truncation lacking the coiled-coil domains
(Atgolgin-84A1-557) were transiently expressed in Nicotiana tabacum and
imaged by confocal microscopy with Airyscan detector. High-resolution
confocal imaging was used to resolve the Golgi cisternae and the ER-Golgi
interface. The data presented here shows that Atgolgin-84A seems to be
localised at a pre-cis-Golgi compartment that is also labelled by one of the
COPII proteins. Optical trapping is a technology in which an infrared laser beam can be
used to capture and manipulate Golgi bodies in planta. The trapping
experiments using optical tweezers revealed differences in Golgi bodies
trapping properties when the truncated version Atgolgin-84A1-557 was
overexpressed in N. tabacum leaves.
Under the hypothesis that Atgolgin-84A could also be implicated in ER
to Golgi trafficking a secretion assay was optimised using Atgolgin-84A as
effector expected to affect the transport of cargo molecules. The trafficking
of cargo was imaged when cells were expressing Atgolgin-84A full-length
and mutant Atgolgin-84A without coiled-coil domains. The cargo was redirected to a different compartment. This hypothesis was also tested using
an α-amylase assay and the secretion index decreased when Atgolgin-84A
was co-expressed with cargo supporting the effect observed using confocal
imaging. The results show strong evidence for a role of Atgolgin-84A in
trafficking between ER and Golgi
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