Reticulons (RTNs) refer to vital proteins that give ER tubules shape.
Endoplasmic reticulum (ER) is a collection of cisternae and tubules that
are experiencing rapid rearrangement. According to recent biological
studies, RTNs take part in many cellular functions such as intracellular
trafficking, apoptosis and cell division. RTNs belong to the reticulons
homology domain (RHD), which is a class of membrane-spanning proteins.
Researchers assert that these cells are present in all the eukaryotic
organisms that they have observed so far. Similarly, the researchers
claim that corresponding proteins are common in endoplasmic reticulum
membranes. The knowledge of the functions of these cells is more
advanced in yeast and animals than in plants. This research hypothesises
that although plant reticulons reconstructs the cortical endoplasmic
reticulum lumen, it does not interfere with intracellular trafficking
between endoplasmic reticulum and Golgi.
This study will investigate whether RTN in plants cells are locally
found in ER cells. Moreover, it will examine whether RTNs do help in
maintaining the physical structure of ER cells, and its possible effect
on transportation of protein materials synthesised by organelles inside
the cell.
Specific aims and milestones
Specific aim and milestones Duration
Membrane spanning proteins 10 hours
Human and yeast cells comparison 12 hours
Cloning animal cells 18 hours
In order to get reticulon plant cells for using in this study, the
researchers cloned coding sequence of RTN using reverse
transcriptase-polymerase chain reaction. They extracted the sample from
Arabidopsistotal seed RNA. Researchers created diverse constructs using
the pVKH18-EN6 binary expression vector. This cloning process created
untagged RTNLB13 as well as, RTNLB13 composed of either N- or
C-terminal Myc tag. Some were merged to yellow fluorescent protein (YFP)
(Sparkes et al 2012, p. 4). Given that, Arabidopsis RTNLB13 has a
canonical dilysine ER retention motif at its C-terminus (KKSE), probably
it comes in touch with cytosol as forecasted (Sparkes et al 2012, p. 5).
Although the C-terminal fusion covers the KKSE motif, it is not apparent
whether this process is essential for retaining ER, considering that
many RTN members in the mammal family does not have similar motif.
Instead, they are found in ER membranes. For determining whether
completely uncovered KKSE recovery progression is obligatory for proper
localisation, the researchers also made constructs with four KKSE codons
at the end attached to the Myc tag.
Expected results
After conducting this experiment, the researchers were expecting to
discover whether reticulon plant cells are locally available in
endoplasmic reticulum (ER). Moreover, this study aimed at proving that
myc-tagged, myc-untagged and YFP-tagged induces extreme physical
alterations on the ER lumen. The study deliberates on proving that
reticulon cells in plants suppress dispersal of soluble proteins ER
lumen. This comes about because it introduces constrictions in the
membrane. In addition, the study will show that this phenotype does not
interfere with the number, shape and dynamics of Golgi organelle, and
synthesis of reporter proteins are not affected.
Alternative outcomes
When RTNs are coexpressed using negative-control (empty) vectors, the
physical structure of the cortical ER is significantly changed (Sparkes
et al 2012, p. 3). This implies that ER tubules will become
undetectable. Instead, a bunch of big vesicle-like structures will
replace them. This means that coexpressing RTNLB13 will induces
destruction of normal cortical structure. Amazingly, this study also
aims at showing that RTNLB13 has no influence on the nuclear envelope.
This result matches the observations that researchers have made on yeast
and animal cells.
Contingency plans
In order to enhance accuracy of this study’s observations, the
researchers compared the behaviour of plant RTNs with the results the
well-known behaviours of yeast and animal cells. Several previous
studies on reticulon cells and their role in intracellular trafficking
between endoplasmic reticulum and Golgi were based on animal and yeast
cells. The researchers used some RTN facts derived from animal cells
when making conclusions on complex plant cells observations.
Ethical considerations
One of the ethical considerations that the researchers considered was
ensuring to seek authorisation of previous studies’ researchers. This
research was solely based on reticulons localised in plant cells, but
the investigators compared their observations with the results acquired
from animal studies when drawing their conclusion. They ensured to
consult their fellow researchers before using their studies. According
to Mazzoni (2002, P. 14), ethical researchers supporting the study
observations using work of other investigators in the related field
should use appropriate references to honour the copyrighted material.
Moreover, a “control experiment” using behaviours of reticulon cells
extracted from human tissue will be mounted. This will help in ensuring
that the conclusions the researchers will make are accurate.
In addition, these researchers conducted some tests on human and yeast
cells, and then related their observations to the plant cells. In order
to avoid violating the human rights of the people whom they used their
cell samples in the study, they ascertained to consult relevant people
and their family members concerning using human tissue on the study.
Moreover, the researchers used cloning method to create RTN plants
cells. In order to enhance accuracy of the study, they drew conclusions
on the functioning of plant reticulon cells they based their
conclusions on the well-known information from yeast and animal cells.
Justification of resources
There is limited research on plant reticulons function hence,
researchers had to compare their outcome to previous studies on animals.
Since these researchers were depending on studies conducted by other
scientists, they needed to seek appropriate permission of these
publishers in order to avoid violating their rights. Identifying some
functions of the cells was challenging, thus the need of hiring three
biologists. The observations and conclusions that were made by the
combined effort of these researchers were more accurate than hiring a
single investigator. Considering that, the study involved comparing the
effect of RTNs in plant cells, and then comparing the results to
conclusions drawn from previous animal and yeast studies, hiring two
plant biologists and one animal cells biologist ensured that the three
individuals could come up with certain conclusions. The head of plant
RTN cells researcher was given the title of Principle Investigator (PI),
and his assistant was titled Co-Investigator (CoI). Similarly, the
animal reticulons cells researcher was called research collaborator
Significance of the study
This study on plant reticulons is crucial to the society since it gives
scientists a base for developing better yielding crop varieties. In the
recent past, climate change has made it essential for botanists to
understand cellular structure of plants in order to develop high
yielding crop varieties. A good understanding of plant cells function
provides a suitable platform for crop engineering (Lee 2012, p. 58). The
society will benefit from this technology since it will manage to grow
short term and high yielding crops.
MAZZONI, C. M. (2002). Ethics and law in biological research. Boston,
Kluwer Academic Publishers.
LEE, H. (2012). Factors regulating endoplasmic reticulum morphology and
quality control. Thesis (Ph.D.)–University of Warwick, 2012.
SPARKES et al. (2012). An Arabidopsis reticulon and the atlastin
homologue RHD3-like2 act together in shaping the tubular endoplasmic
reticulum. New Phytologist. Wiley Online Library. 197(2).