Small Darts and Circles, Large Hairpins:

The Relationship Between Size and

Morphology in Arabidopsis Worms.
Authors: Jessie MNG Lopez, Andrew J Hill, and Ernest Kwok
Introduction
In 2000, a group of scientists demonstrated a groundbreaking technique for
visualizing proteins in arabidopsis.(1) Cutler, et al, created a library of arabidopsis strains
that bear GFP at different places in their cDNA. Some of these transgenic strains, reveal
strange shapes under fluorescence, seemingly unmappable to previously known
subcellular structures. One of the these, pEGAD::cDNA 739-2, exhibits glowing, usually
curved, shapes referred to as worms. These worms come in various morphologies, and
in various sizes. A single cell could have many, few or no worms. Their shape, size, and
placement is mysterious. We wanted to see if a relationship exists between the type of
shape of the worms and the size of the worms.
Materials and Methods
The strain of transgenic pEGAD::cDNA 739-2 dart seedlings we used were grown
on MS media for six days in Dr. Kwoks laboratory. The seedlings were grown in the dark.
All images were of the hypocotyl.
All z-stack images were taken on a Zeiss Observer Z1 63x oil immersion planAPOCHROMAT objective, with numerical aperture of 1.4, with the fluorescent bulb at the
lowest setting. All 2-D images were taken on a Zeiss Axio Imager A2 100x oil immersion
EC Plan-NEOFLUAR objective, with numerical aperture of 1.3. We used a GFP filter set
and the fluorescent light attenuated to 60%.
Measurements were taken using Zen softwares spline trace function. Data
was organized, and averaged in Excel. Statistics were performed using InStat software.
Scale bars were put on in Photoshop, no images were otherwise manipulated.
A.

B.

C.

D.

E.

F.

Figure 1. Distinct
Morphologies Worm shapes
were categorized into six
mutually exclusive categories:
A. Thetas, B. Hairpins, C.
Circles, D. Darts, E. Figure
Eights, and F. Double
Hairpins. The last group, G.
Miscellaneous, were not
included.
G.
All
scale
bars
A. represent
10 m.

Results
Different Shapes are Different Morphologies
Without knowing anything about the structure or function of these proteins other
than what we could guess by looking at them, we were initially at a loss. These worms
exhibited a variety of strange morphologies that didnt look like any other organelles or
structures weve learned to expect in a cell. Some appeared as rings, some were wiggly
lines, some were figure eights, some were other shapes entirely. How could we hope to
define the relationship between size and shape when we didnt even understand their
shape alone?
Initially we hypothesized that perhaps they were actually all circles, just viewed at
different angles that made them look like the various shapes we were seeing. We used
rubber bands to model distorted circles, viewed them edge on and were able to reproduce
all the shapes we were seeing just by twisting and assuming that part of the structure was
out of the plane of focus. Therefore our first step was to take z-stack images and see if
perhaps these all had the same essential morphology: circle. Those 63x oil immersion zstack images convinced us that these were in fact different morphologies and not just
circles distorted and viewed at awkward angles. We made sure to get at least one or two
worms in clear sharp focus and took deep stacks to give ourselves room to view above
and below the worms of interest. Upon review of the images it was clear that worms of
various morphologies that were bright and sharp in the plane of focus did not have
significant extensions above or below the plane of focus. The software that we were using
didnt have the ability to eliminate the out of focus blur, but both of us were convinced that
there are in fact different worm morphologies, and we could proceed with two-dimensional
analysis without worrying that we were missing significant 3-D information.
Categories of Morphologies are Mutually
Exclusive and Jointly Non-exhaustive
We decided on several morphological
categories: circle, figure eight, theta, hairpin, and
double hairpin (see Fig. 1) and made sure to take
many images of each morphology, in each seedling.
We excluded all morphologies that did not fall into
these categories. All of the worms in one seedling
were larger, on average, than all of the worms in
another seedling and we wanted to eliminate the
variation due to individual seedlings, so we used
matched data, with equal numbers of measurements
from each of two seedlings. For size measurements
we used the spline curve function on Zeisss Zen
software. As shown in Fig. 2, we traced through
the middle, along the curved path length, of each
worm, then moved the traced line off to the side, and
traced again. There were small unavoidable
variations in the measurements we took, so we
traced each individual worm three times and
averaged to get a more accurate final measurement.
The data were gathered in an Excel file, organized
by seedling and morphology.

Figure 2. Measuring the

Worm Path Length.
Screenshot of the spline tool on
the Zen software. We averaged at
least three measurements to
account for subtle variations in
spline placement.

Many worms we saw did not fall into one of our six neat morphological categories.
Many, like the worm labeled G. in Fig. 1, were too complex and unclassifiable, and were
therefore not included in our data analysis.

Figure 4.
Median
lengths in
ascending
order
The median
lengths of each
morphology listed
in ascending order
as well as the
number of data
points included in
analysis. Bars
show standard
deviation.
N= 12

N= 12

N= 14

N= 10

N= 12

N= 10

There are statistically significant differences in median size between different

worm morphologies.
The worms that fell within our classification groups showed several significant
differences between groups. Figure 4 summarizes the median lengths of the
morphological categories and shows the trend, from the smallest shapes, dart and
circles, up to the largest shapes, hairpins and double hairpins.
When we applied Kruskal-Wallis non-parametric statistical analysis, we found
that there was a statistically very significant difference between the smallest shapes
and the largest shapes. Table 1 gives the significant p values from our statistical
analysis.

Table 1.
Summary of
p values
of statistically
significant size
differences
between categories
of worm
morphology.

Mean Rank !
Comparison
!
==================================
!
hairpins vs. circles
!
hairpins vs. darts
!
figure 8s vs. darts
!
circles vs. 2x hairpins
!
2x hairpins vs. darts
!
thetas vs. darts
!

P value !
=============!
** P<0.01!
*** P<0.001!
** P<0.01!
*** P<0.001!
*** P<0.001!
*
P<0.05!

Discussion/Conclusion:
My conception of the morphology of
these structures has evolved over the course of
this investigation. From initially hypothesizing
that they are all thick singular circular loops
twisted into various shapes, we now believe
these worms are composed of many thin
filaments, all stuck to each other appearing to
our eyes as aggregates. Figure 5 is an
example of the kinds of wispy multi-looped
shapes that eventually led to this assessment.
Figure 5. A single worm
Kwok, et al, at CSUN has undertaken to
of miscellaneous
understand these worm shapes. In personal
morphology. These types of
conversation, he has told us that the protein
uncategorizable worms appear
GFP is attached to in this strain belongs in the
to be made of aggregates of thin
annexin family. The annexin family of proteins
filaments.
are involved in a variety of tasks, including
vesicle transport, interaction with phospholipids at the plasma membrane, aggregation,
and both endocytosis and exocytosis. This versatile calcium binding protein family
often is involved in binding tasks, perhaps we are seeing how it binds to itself,
clustering thin filaments into visible clumps.
Many subcellular structures in the body, such as collagen and elastin self-bind,
in, respectively, either an organized or disorganized fashion. Perhaps worms are
made of a somewhat disorganized self-binding annexin filament. If that is the case,
then could the self-binding domains be disrupted, perhaps through changing the Ca2+
levels or by a protease of some kind? If the cross filament bonds could be disrupted
then the individual strands that make up the aggregate could be visualized separately,
like spaghetti strands that have clumped together while cooking and are freed by olive
oil.
Why are darts smaller than hairpins? Do worms all start out as simple small
shapes and grow to larger shapes, eventually becoming tangles of partially aggregated
fibers? Future inquiry could compare seedlings of different ages, to see if larger more
complex shapes are found in older seedlings, or perhaps involve a long time lapse (a
couple days) showing how a single worm develops over time.

1. Random GFPcDNA fusions enable visualizaFon of subcellular structures in cells of Arabidopsis

at a high frequency
Sean R. Cutler, David W. Ehrhardt, Joel S. GriOs, and Chris R. Somerville
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3718-23.