report2.tex

% -*- mode: LaTeX; LaTeX-command: "latex -shell-escape" -*-

% Mike's instructions for this report:

% Just to assist you in finishing your final lab report, Sarah and I
% are including some guidelines below:

% 1. This report focuses on motility.  It should cover random
%    motility, under agarose folate chemotaxis, and development.

% 2. We are interested in you quantifying different aspects of
%    motility under the different conditions, such as speed of cells,
%    persistence, etc.  At least 2 different aspects of motility
%    should be quantified for cells moving randomly and
%    chemotactically.  Those that go beyond this minimum requirement
%    will of course be rewarded!

% 3. Development was a big experiment all by itself.  Remember that
%    we did under agarose development, over agarose development, and
%    development on bacterial lawns.  You saw the greatest differences
%    between wild-type and mutant cells in the over agarose and
%    bacterial lawn experiments, so those should certinly be
%    addressed.  In the under agarose experiment many of you
%    successfully looked at individual fluorescent cells moving in
%    streams.  Should you choose to analyze and quantify that
%    motility, that will be considered a major bonus.

% 4. Finally, remember the big, overall questions - how do the
%    mutant and wild-type cells compare to each other, and how do the
%    different motility assays compare to each other?  Keep those in
%    mind as you write your discussions.

% Remember that if your websites are well done, you've already done
% most of the work for this report.  You are now just tying it all
% together.

% http://homepages.uconn.edu/~mb2225vc/MCB_2225/Cellbio6_files/Cellbio6/Cell_Motility_1.html

\documentclass[12pt]{article}

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\usepackage{authblk}            % For \author{} and \affil{}.
\usepackage{paralist}           % In-paragraph list, \inparaenum{}.
\usepackage{xspace}             % Add trailing macro space.
\usepackage{datatool}           % Import csv file as table.
\usepackage{natbib}             % For bibliography{}.
\usepackage[nottoc,numbib]{tocbibind} % Enumerate bibliography section.
\usepackage{url}                % For processing url{} in .bib file.
\usepackage{graphicx}           % Figures.
\usepackage{minted}             % Python source code with highlighting.
\usepackage{epstopdf}           % Convert .TIF to .PNG
\usepackage{textgreek}          % Non-italic micro symbol.
\usepackage{gensymb}            % For \degree C.

% Package options
\epstopdfDeclareGraphicsRule{.tif}{png}{.png}{convert #1 \OutputFile}
\AppendGraphicsExtensions{.tif}

% Convenience macros
\newcommand{\dicty}{\emph{Dictyostelium discoideum}\xspace}
\newcommand{\ul}{~\textmu l\xspace}

\title{\vspace{-4.5ex} Myosin II in \dicty is required for slug social development, but not for chemotaxis}
\author[1]{Pariksheet Nanda}
\author[2]{Dinkar Ahuja}
\affil[1]{Department of Cell Biology}
\affil[2]{Department of Molecular and Cell Biology}
\date{April 20, 2015}

\begin{document}

\maketitle{}

\begin{abstract}
  \noindent
  The myosin II motor protein helps orient motility of \dicty.
  Cell stray from their intended direction without its involvement, but yet the cell maintains other measurable characteristics of velocity, and persistence.
  Directionality is expected to be worse, but cannot be differentiated using simple statistical methods.
  Myosin II also plays a critical role in \dicty slug social development, as a myosin II null mutant, HK321, dissociates after the mound stage instead of coalescing into slug formation.
  In this report, we observe \dicty motility during their
  \begin{inparaenum}[\itshape a\upshape)]
  \item vegetative cycle experiencing random motility,
  \item under agarose using folate chemotaxis,
  \item and during their social development cycle.
  \end{inparaenum}
\end{abstract}

\section{Introduction}
\label{sec:intro}

\subsection{Background}
\label{sec:bg}
Axenic \dicty cells in their vegetative growth state move randomly.
Chemotaxis is cell movement, or motility, towards a chemoattractant.
Cells can sense as little as a 1\% difference in chemical concentration between different parts of the membrane and move toward the source of higher concentration.
In the case of \dicty, two chemoattractants are folate and cyclic AMP (cAMP).
The former chemical, folate, is thought to be emitted by the organism's food sources, such as bacteria.
The latter, cAMP, is produced by \dicty itself to signal aggregation during the social development cycle.

\subsection{Glossary}
\label{sec:glossary}
\begin{description}
\item[Adhesion]
  Attraction between dissimilar molecules.
  Contrasts with cohesion, where the attraction is between identical molecules.
\item[Cytoskeleton]
  Protein fiber network in the cytoplasm involved in cell shape, support, locomotion, and transport of materials within the cell.
\item[Dictyostelium discoideum]
  An amoeba used as a model organism.
\item[Myosin II]
  Motor protein which uses ATP energy to move actin filaments for contraction, cytokinesis, and vesicle transport.

\item[Chemotaxis] 
  Cell movement towards, or away from, a chemical compound.
\item[Folate] 
  Chemotactic chemical known to attract \dicty.

\item[Vegetative cycle]
  Cell growth and division, under ideal \emph{semi-log phase} conditions.
  Graphing cell density, during ideal conditions, on a base-2 logarithmic axis, yields a linear growth region, which is known as the \emph{log phase} of cell growth.
  The ``semi-'' in the semi-log term mentioned earlier is in reference to the contrasting, linear time axis.
\item[Social development cycle]
  Cell migration to form aggregation bodies, under stressful starvation conditions.
  Wild type \dicty form slugs, then mounds, and finally fruiting bodies to spread spores to distant locations.

\item[Motility]
  Full cell body movement.
\item[Persistence]
  The ratio of distance traveled to displacement travelled; a unitless number between 0 and 1.
  When measuring a given cell, average all such measurements.
\item[Directionality] 
  The cosine of the angle subtended between the instantaneous and final positions; a unitless number between -1 and 1.
  When measuring a given cell, average the absolute values of all such measurements.

\item[Fluorescence]
  A high contrast imaging technique for studying biological processes.
\end{description}

\subsection{Hypotheses}
\label{sec:hypo}
\begin{enumerate}
\item Vegetative cells have random motility.
  On average there is low directionality and low persistence.
\item Cells without Myosin II will move slower than wild-type cells,
  since the motor protein plays a role in contraction.
\end{enumerate}

\subsection{Null Hypotheses}
\label{sec:nullhypo}
\begin{enumerate}
\item Vegetative cells have comparable measures motility in speed, directionality and persistence as chemotactic cells.
\item Cells without Myosin II will behave the same as wild-type cells.
\end{enumerate}

\subsection{Objective}
\label{sec:exp}
Quantify the motility of two cell lines: NC4A2, and HK321; the latter being the Myosin II null mutant.
Observe movement in their vegetative/growth state, and in a folate chemotactic gradient.
Compare motility with social development induced by starvation.

\section{Results}
\label{sec:res}
The under- and over-agarose experiments, as well as bacterial plate growth experiment all demonstrate that the HK321 cells form smaller mounds than the wild type.
HK321 never progresses beyond mound aggregation into slug or fruiting body formation as evidenced by the long term bacterial plate experiment in Fig.~\ref{fig:bacterial}.
Additionally, the mounds would disintegrate at the 16 hour mark instead of advancing to the next stage of development as captured in Video 2 of our experiment blog.
We counted a total of 14 slugs with our NC4A2 cell line during the over agarose assay shown in Fig.~\ref{fig:dev}, but no slugs with our HK321 cells.
As the bacterial plates were not under constant observation, we observed magnitudes more fruiting bodies than slugs, indicating most of the slugs had converted to fruiting bodies.

To understand other ways in which the HK321 organization differed in preventing slug formation, we analyzed its chemotactic movement to the wild type under agarose.
Surprisingly, it moved faster and had higher directionality.
However movement along the chemical gradient axis was lower, as evidenced by the skewness when examining the X-coordinate histogram in Fig.~\ref{fig:xhist}.
Skewness of close to 0 should correspond to random motility, whereas the HK321 was between 0 and the higher, nearly 2 skewness, of the wild type.

We were not able to present data of HK321 motility during development due to non-availability of fluorescent HK321 cells.
The HK321 cell line was found to be faulty right before the development experiment, and the new batch had few transfected cells (4-9\% yield) which did not produce any tracks.

\begin{figure}\centering
  \includegraphics[width=0.8\textwidth]{data/figj_exp8_bacterial_plates/FullResolution_figj_exp8_bacterial_plates.tif}
  \caption{Bacterial lawn development of NC4A2 and HK321.
(A) Low concentration plate of NC4A2 showing circular spread on bacterial lawn.
(B) Low and (C) high concentration, top-down, zoomed photos of NC4A2 plate showing fruiting bodies.
(D) Low and (E) high concentration plates of HK321 showing characteristic annulus aggregations.
}
\label{fig:bacterial}
\end{figure}

\begin{figure}\centering
  \includegraphics[width=0.8\textwidth]{data/figj_exp8_nc4a2_dev/FullResolution_figj_exp8_nc4a2_dev.tif}
  \caption{Over agarose NC4A2 development.
(A) Vertical photo of two fruiting bodies.
Scale bar applies to foreground fruiting body.
(B), (C) Example top-down photos of slugs and fruiting bodies.
In total 14 slugs were found.
}
\label{fig:dev}
\end{figure}

\begin{figure}\centering
  \includegraphics[height=0.89\textheight]{fig/exp3_exp7_rose_plots.eps}
  \caption{Rose plot of cell tracks.
The chemokinetic chemical, folate, was placed on right side of the cells.
The dashed center line indicates $x=0$.
(Top) Random motility of NC4A2, but it was not an ideal control since it was not under agarose.
(Center) HK321 shows some movement toward the chemokinetic chemical, but was poorly directed and also moved a fair distance along the y-axis.
(Bottom) NC4A2 cells showing strong movement toward folate on the right.
}
\label{fig:rose}
\end{figure}

\begin{figure}\centering
  \includegraphics[width=\textwidth]{fig/exp3_exp7_x_histogram.eps}
  \caption{Histograms of cell x-coordinates corresponding to Fig.~\ref{fig:rose}.
Ideally these histograms would have been placed alongside Fig.~\ref{fig:rose}, but there was not enough space.
Note that $n$ here refers to the number of x-coordinates which is more than the number of $tracks$ in Fig.~\ref{fig:rose}.
(Top) Random motility of NC4A2 showing low skewness of -0.3.
(Center) HK321 chemotaxis shows some skewness of 1.4.
(Bottom) NC4A2 chemotaxis showing the most skewness of nearly 2.
}
\label{fig:xhist}
\end{figure}

\begin{figure}\centering
  \includegraphics[width=\textwidth]{fig/exp3_exp7_exp10_metrics.eps}
  \caption{Histograms of 3 metrics for random movement, chemotaxis, and development.
(Left column) At face value, directionality appears similar in most cases, but one might be able to better differentiate them by comparing the lower and higher percentiles.
(Center column) Persistence is broadly distributed across the 0-1 range in random movement.
Persistence is improved in chemokinetic conditions.
(Right column) Velocities in the 4 situations look fairly similar.
Assuming cells being under agarose affects their velocity, we should compare only the top and bottom histograms with eachother since they were both over agarose, and the middle two histograms since they were both under agarose.
}
\label{fig:metrics}
\end{figure}

\section{Discussion}
\label{sec:dis}
The HK321 cells not being able to advance to the slug formation stage may have been caused by too few cells reaching the mounds to achieve cell critical mass (of about 10,000 cells).
Missing this critical number, in turn, may be due to lower HK321 directionality which, as shown in Fig.~\ref{fig:metrics}, more closely matches random motility than wild type chemotaxis.
In other respects of speed and persistence, HK321 is comparable to NC4A2.
The reason that a critical number exists assumes that the development feedback mechanism is time limited.
In other words, cells do not keep aggregating until critical mass.
The time duration of mound formation in HK321 vs NC4A2 videos is on the order of 10 hours from start to finish, and about 2 hours during rapid aggregation.
Another possible reason for not advancing to the slug stage could be the myosin II motor protein playing a more direct role at this development stage;
perhaps the contractile forces from stress fibers play a more direct role in spatial organization or checkpoints for slug formation.

In the bacterial growth plate experiment, there were a low number of slugs and a high number of fruiting bodies with the wild type.
This could either mean that conditions for fruiting body development were optimal for a high conversion ratio from slugs to fruiting bodies.
This was surprising since the plates were kept in the incubator and away from light.
In total, many more slugs were counted with the over agarose development.
This may have been due to 

It was surprising to see the mean velocity under agarose higher than over agarose.
We expected that the under agarose assay would skew the speed measurement, and possibly also directionality, and persistance.
Motile cells under agarose might move slowly due to their cytoskeletons working against the viscosity and weight of the agarose.
Besides the effect on speed, under agarose may also show an artificial improvement in directionality and persistence as the cell would be able to process more signal events per unit distance.
The higher speed in chemokinetic conditions may indicate the cells expend more energy in motility for the possibility of finding food.
It might also indicate a higher concentration gradient, and that concentration regulates speed.

The directionality data presented in Fig.~\ref{fig:metrics} for development has a source of systemic error as it does not account for the spiral movement streams.
When directly taking the cosine of the cell movement angle, perfect directionality corresponds to the cell moving towards the right side of the camera field of view along a chemotactic gradient.
Thus the directionality measurement models a linear path, whereas the cyclic AMP (cAMP) wave is propagated by cells along curved tracks.
A more accurate measurement would be to account for these curved paths in the directionality equation itself.
This could be done using the tracking data alone by measuring colocalization of cells that might move along the same track, fitting a curve to the path, and using the normal to this path as the directional reference at each point.
Of course, one must then also go back and evaluate and apply such an equation with the linear chemotaxis data to not introduce bias from different analyses of the same measurement.

\section{Materials and Methods}
\label{sec:matmeth}
HK321 is a Myosin II heavy chain null mutant derived from NC4A2 \citep{CM:CM5}.
HL5 media, treated with antibiotics, was used to feed the cells.
The antibiotics protect the media from bacterial, but not fungal, contamination.
Plates used were standard 60 mm plastic cell culture dishes and were seeded with 10 ml of media and a $5 \times 10^4$ cells/ml titer.
The cells counting protocol is listed in Supplement~\ref{sec:proto:celldens}.

Microscopy videos were captured using the Micro-Manager software \citep{ausubel_computer_2010,JBM36} version 1.4.19 controlling a Scion CFW-1310M CCD detector on a manual Nikon Ti inverted microscope.
Thorlabs TLED devices provided both, diascopic and epifluorescence, illumination modalities.
An Arduino microcontroller, controlled by Micro-Manager, TTL triggered the LEDs.
The configuration file is included in Supplement \ref{sec:cfg}.

Data analysis was done using Python scripts run inside an IPython shell \citep{PER-GRA:2007} on Gentoo GNU/Linux \citep{stallman_gnu_2014}.
Cell tracking was done using the TrackMate plugin of FIJI \citep{schindelin_fiji:_2012}, and images were arranged using the FigureJ plugin of FIJI.
Graphs were created using Matplotlib \citep{Hunter:2007}.
This manuscript was prepared using \LaTeX{} with the GNU~Emacs editor and its accompanying AUCTex package.
All source files are freely available on GitHub (\url{https://github.uconn.edu/pan14001/cell-bio-lab-2015}), and have been released into the public domain.
The scripts used to generate the graph has also been included in Supplement~\ref{sec:code} as well as GitHub.

\bibliographystyle{biochem}
\bibliography{report1}

\section{Supplements}
\label{sec:sup}

\subsection{Protocols}
\label{sec:proto}

\subsubsection{Over agarose development}
\label{sec:proto:overdev}
\begin{enumerate}
\item Prepare solution with 10$^{\text{7}}$ stock (non-fluorescent) NC4A2 or HK321 cells, depending on the experiment.
  Add 5\% NC4A2 (GFP) and HK321 (RFP) cells.
  \begin{enumerate}
  \item Centrifuge for 5~minutes at 500$\times$g to remove original media.
    Discard HL5 media, add 5~ml SorNC and vortex.
  \item Centrifuge for 5~minutes at 500$\times$g to remove impure SorNC.
    Discard SorNC buffer, add 2ml SorNCs and vortex.
  \item Break up cell clumps.
    Use pipetteman 20 times to suck and spit out cells in test tube to reduce clumping.
  \end{enumerate}
\item Create agarose p60 plate by pipetting 1.5\% agar solution and waiting about 15 minutes for it to solidify.
\item Adhere cells on dish.  
  Pour the 2~ml of the cells in SorNC and spread onto the the dried agar of the p60 dish.
  Allow 1~hour to settle, as cells hardly stick to agar.
  Remove buffer from side of dish with p1000, and blot media with rolled up Kim wipe
\item Use video microscopy using 4x objective.
  Setup transmitted light channel with low camera exposure, otherwise accumulation will saturate the camera.
  Acquire images for at least 30~hours.
\item Snap image with macroscope when slugs and fruiting bodies develop.
\end{enumerate}

\subsubsection{Under agarose development}
\label{sec:proto:underdev}
\begin{enumerate}
\item Prepare solution with 10$^{\text{7}}$ stock (non-fluorescent) NC4A2 or HK321 cells, depending on the experiment.
  Add 5\% NC4A2 (GFP) and HK321 (RFP) cells.
  \begin{enumerate}
  \item Centrifuge for 5~minutes at 500$\times$g to remove original media.
    Discard HL5 media, add 5~ml SorNC and vortex.
  \item Centrifuge for 5~minutes at 500$\times$g to remove impure SorNC.
    Discard SorNC buffer, add 5~ml SorNCs and vortex.
  \item Break up cell clumps.
    Use pipetteman 20 times to suck and spit out cells in test tube to reduce clumping.
  \end{enumerate}
\item Create agarose p60 plate by pipetting 5~ml of 1.5\% agar solution and waiting about 15 minutes for it to solidify.
  Cut a small wedge of the agar at the outer edge and remove the wedge.
\item Adhere cells on dish.  
  Pour the 5~ml of the cells in SorNC into the p60 dish.
  Allow 30~minutes to settle.
\item Remove the agarose sheet with a spatula and place on the dish with cells.
  Remove buffer from wedge cut side of the dish with p1000, and blot media with rolled up Kim wipe
\item Wait 4-6 hours and begin video microscopy using 4x objective.
  Setup transmitted light channel with low camera exposure, otherwise accumulation will saturate the camera.
  Acquire images for at least 30~hours.
\end{enumerate}

\subsubsection{Under agarose chemotaxis}
\label{sec:proto:chemo}
\begin{enumerate}
\item Prepare 100\ul of cells at $500 \times 10^{4}$ cells/ml for each of the two troughs, or $100 \times 10^{4}$ cells in total.
  \begin{enumerate}
  \item Put cells in 15~ml tube.
  \item Centrifuge cells for 5~min at 1000~rpm at 4~\degree C.
  \item Discard HL5 media.
  \item Resuspend in Sorenson's buffer (Sor) and recentrifuge.
  \item Discard Sor.
  \item Resuspend in SM media.
  \end{enumerate}
\item Create 2 agarose plates (Fisher $60\times15$~mm):
  \begin{enumerate}
  \item Create 1.5\% or 0.5~g/33~ml agar in 33~ml SM media.
  \item Melt agarose/SM in 125~ml flask in the microwave in 6 second bursts with the flask cap loose.
  \item Add 8~ml agarose per plate.
  \item Allow 15~minutes to cool.
  \item One agarose plate is for practise.
    Use the half-size dissection razor blade to cut the center trough and 2 outer troughs.
  \end{enumerate}
\item Fill the plate: each of the outer troughs with 100\ul of cells in SM, and  folate to the center trough.
\item After 6~hours, image each dish with transmitted light.
\end{enumerate}


\subsubsection{Cell density measurement}
\label{sec:proto:celldens}
\begin{enumerate}
\item Putting adhered cells back into suspension, requires agitation.
  The agitation method depends on the container; for cells in a glass conical flask, a bacterial shaker is used to keep the cells moving and prevent settling.
  For flasks and test tubes that have been sitting and require agitation:
  \begin{enumerate}
  \item Make sure the container cap is  to screwed on tight.
  \item Press the container on a bench top vortex mixer for a few seconds at a time.
  \item Release the tension on the screw cap to prevent suffocation.
  \end{enumerate}
  For cells on a plate, we cannot shake the container as nothing secures media from spilling out of the low wall height.
  For growth plates only:
  \begin{enumerate}
  \item Agitate, or \emph{titurate}, plate bottom using 5 ml pipette.
  \item Pipette out a few ml of media using a Pipettman, and release the media back across the bottom of the plate at maximum velocity, but do not empty all liquid as that would blow in bubbles.
  \item Keep the pipette mouth perpendicular and move it around regions of the bottom surface to clear areas of adhered amoebae.
  \item Verify tituration was effective by viewing plate bottom using 10x lens in microscope.
  \item Retiturate until satisfactory.
  \end{enumerate}
\item Pipette 10\ul of titurated solution onto hemocytometer.
\item Count at least 100 cells, or count all etched squares using mechanical counter.
\item Clean the hemocytometer and coverslip with de-ionized water.
  Use bench top microscope to confirm they all cells have been washed away from the grid areas.
  This inspection is especially important to prevent errors when measuring low cell densities.
\end{enumerate}

\subsection{Figure Source Code}
\label{sec:code}

\subsubsection{Rose plots in Figure~\ref{fig:rose}, and Histograms in Figures~\ref{fig:xhist} and \ref{fig:metrics}}
\label{sec:code:rose}
\inputminted[linenos]{python}{fig/exp3_exp7_rose_plots_with_x_histogram.py}

\subsubsection{FIJI TrackMate XML File Reader used by Supplement~\ref{sec:code:rose}}
\label{sec:code:trackmate}
\inputminted[linenos]{python}{fig/trackmate.py}

\subsection{Software Configuration}
\label{sec:cfg}

\subsubsection{Differences with Micro-Manager Configuration File in  Report 1}
\label{sec:cfg:mmdiff}
\inputminted[linenos]{diff}{data/exp10_scion-tled.cfg.diff}

\end{document}