Observation and quantification of protein production in single living cells

171 Pages·2017·5.36 MB·English

by  Ibrahim Kays

Observation and quantification of protein production
in single living cells

by

Ibrahim Kays

Integrated Program in Neuroscience
McGill University, Montréal
November, 2016

A thesis submitted to McGill University in partial fulfilment of the requiremen ts of
the degree of Doctor of Philosophy
© Ibrahim Kays, 2016
Abstract
Accurate quantification of protein production is fundamental to understanding basic
molecular and cellular processes. Dysregulation of protein levels can harm cells and lead to
diseases such as cancer and neurodegenerative diseases. However, little is known about how
protein production in a cell changes over time and in response to external factors. The current
assays used to quantify protein production are invasive, time consumin,g and have poor
resolution. As a result, researchers have turned to mRNA expression as a measure for protein
abundance, although this has been demonstrated to be inaccurate .
To address these issues, my thesis explores new tools and techniques Id eveloped to
monitor protein production in single living cells. By simultaneously examining the levels of
mRNA and protein of a gene from a single cell, I describe a system used to determine how
individual cells vary in their transcriptional and translational landscapes, and demonstrate the
low predictive power of mRNA levels over protein abundance.
My second approach to understand protein production is aimed at directly observing
protein synthesis in living cells. I describe the generation of animals used for imaging protein
production in single cells in real time. I also describe a system that uses the reconstitution of split
GFP as a spatial and temporal quantitative marker of local protein synthesis. I used single-cell
quantitative imaging, electrophysiology and immunocytochemistry to demonstrate that proteins
produced with split GFP reporters function properly, and that their level of production correlates
with the intensity of the reconstituted GFP signal.
The experiments presented in this thesis demonstrate tools I developed to probe protein
production with high spatial and temporal resolution. The tools and reagents are accessible to a
wide range of researchers and the assays provide high accuracy and reliability. Protein analysis
in single cells can reveal unprecedented insight into the dynamics of the gene expression.
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Résumé
La quantification précise de la production de protéines est un outil fondamental pour
comprendre les processus cellulaires et moléculaires de base. La dérégulation des niveaux de
protéines peut endommager les cellules et mener à des maladies telles que le cancer et les
maladies neurodégénératives. Cependant nous en savons très peu sur la façon dont la production
de protéine change dans une cellule avec le temps ou en réponse à des facteurs externes. Les
méthodes actuelles de quantification de la production de protéines sont invasives, prennent
beaucoup de temps et ont une mauvaise résolution. En conséquence les chercheurs se sont
tournés vers l'expression de l'ARNm afin de mesurer l'abondance de protéines, bien qu'il ait été
démontré que cette méthode est imprécise.
En réponse à ce problème, mon travail de thèse explore de nouveaux outils et techniques
que j'ai développés afin de mesurer la production de protéines dans une cellule vivante. En
examinant simultanément les niveaux d'ARNm et de protéines pour un gène dans une cellule
unique, je décris un système qui peut être utilisé pour déterminer les variations de cellule à
cellule dans les paysages transcriptionel et traductionel, et je démontre la capacité faible du
niveau l'ARNm à prédire l'abondance de protéines.
Ma seconde approche visant à comprendre la production de protéines et dirigée
directement vers l'observation de la synthèse de protéines dans des cellules vivantes. Je décris
l’élaboration d'animaux utilisés pour capturer la production de protéines dans une cellule unique
en temps réel. Je décris aussi un système qui utilise la reconstitution d'une protéine fluorescente
verte (PFV) découpée comme marqueur quantitatif spatial et temporel de la synthèse locale de
protéines. J'ai utilisé de l'imagerie quantitative de cellules uniques, de l'électrophysiologie et de
l'immunocytochimie afin de démontrer que les protéines produites avec comme reporter la PFV
découpée fonctionnent correctement, et que leur niveau de production sont en corrélation avec
l'intensité du signal de la PFV reconstituée.
Les expériences présentées dans cette thèse démontrent les outils que j'ai développés afin
d'examiner la production de protéines avec une haute résolution spatiale et temporelle. Les outils
et réactifs chimiques sont accessibles à une grande variété de chercheurs, et cette méthode
fournit une haute précision et fiabilité. L'analyse de protéines dans des cellules uniques peut
révéler une connaissance approfondie sans précédent de la dynamique de l'expression génique.
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Acknowledgements
My experience in Dr. Brian Chen’s laboratory has been nothing short of transformative. I
was an undergraduate with no research experience when Brian provided me an opport unity to
join his team. He patiently guided me through this journey, helping develop my skills both in the
lab and outside along the way. My gratitude also goes to Drs. Don Van Meyel, David Stellwagen
and Keith Murai for invaluable advice and guidance , as well as to members of my advisory
committee Drs. Artur Kania and Hiroshi Tsuda.
I would like to thank all my colleagues at the Centre for Research in Neurosciences. A
special thank you to Drs. Tiago Ferreira, Emily Peco, Todd Farmer and Haider Al -Timimi for
hallway chats that taught me more lessons than seminars. To Sejal Davla for constantly sharing
her expertise in everything from fly food to Indian food. Many thanks to Chris Salmon, Benny
Kacerovsky and Dr. Gael Quesseveur for help with mouse work . I will always cherish the
memories of the breakfasts I had every Thursday with Hunter Shaw, charmer of the sixth floor
volunteers.
I am indebted to Dr. Chiu -An Lo, who watched and helped me grow since my undergraduate
years, and together with Tsung -Jung Lin t aught me most of what I know about molecular
biology. A special thank you to Dr. Farida Emran, the jane of all trades who also coached and
helped me with every aspect of my graduate work.
Last but certainly not least, to my guide and co -conspirator Dr. Ve drana Cvetkovska, you
have helped realize this work in more ways than you know. I dedicate this thesis to my parents
Dima and Anwar, my sister Yasmina and my pug Winston, for your unconditional love , support
and prodding.

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Contribution of authors
This thesis is presented in thesis-based format in accordance with McGill University
Graduate and Postdoctoral Studies guidelines. It comprises original work from one published
manuscript, one submitted manuscript and one currently in preparation.

The work in this thesis is based on the Protein Quantification Ratioing (PQR) technique
that I co-developed with Dr. Chiu-An Lo, published as:
Lo C*, Kays I*, Emran F, Lin T-J, Cvetkovska V, Chen BE. Quantification of Protein Levels in
Single Living Cells. Cell Reports. 2015;13(11):2634-2644. doi:10.1016/j.celrep.2015.11.048.

Brian E. Chen designed the experiments and supervised the project. Chiu-An Lo, Ibrahim Kays,
Farida Emran, Tsung-Jung Lin, Vedrana Cvetkovska and Brian E. Chen performed experiments
and analyzed the data. Chiu-An Lo, Ibrahim Kays, and Brian E. Chen wrote the manuscript.

The published technique, to which I contributed 4 years of my graduate work, constitutes Dr.
Lo’s PhD thesis work, obtained under Dr. Brian Chen in 2016, and its development and
validation are outside of the scope of my thesis. In this thesis I use our published technique,
described throughout the thesis and in detail in Chapter 1, as a stepping stone for the
development of novel systems and techniques. As per the McGill University Graduate and
Postdoctoral Studies guidelines, I have obtained written consent from Dr. Lo to describe the
technique and include it as a resource for my work.

A modified version of Chapter 2 has been submitted for publication as:
Kays I, and Chen BE. Protein and RNA quantification in single cells, submitted

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Ibrahim Kays and Brian Chen designed the experiments. Ibrahim Kays collected and analyzed all
the data. Ibrahim Kays wrote the manuscript. Brian Chen supervised the study.

A modified version of Chapter 3 is in preparation for publication as:
Kays I, and Chen BE. Direct observation of local protein synthesis in vivo, submitted

Ibrahim Kays and Brian Chen designed the experiments. Ibrahim Kays collected and analyzed all
the data. Ibrahim Kays drafted the manuscript. Brian Chen supervised the study.

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Table of contents
Abstract .......................................................................................................................................... ii
Résumé .......................................................................................................................................... iii
Acknowledgements ...................................................................................................................... iv
Contribution of authors ................................................................................................................ v
Table of contents ......................................................................................................................... vii
List of figures ................................................................................................................................. x
List of abbreviations .................................................................................................................... xi
Chapter I - Current state of quantification of protein production ......................................... 1
1.1 Introduction ....................................................................................................................... .2
1.2 Protein structure ................................................................................................................. 3
1.2.1 The primary structure of a protein is its linear chai .n.................................................... 4
1.2.2 Secondary structures interconnect and stabilize protein residue s................................. 5
1.2.3 Protein folding and maturation are prerequisite to functio .n......................................... 6
1.3 Protein function ................................................................................................................ 11
1.3.1 Regulation of protein function .................................................................................... 11
1.3.2 Regulated protein synthesis and degradation ............................................................. .12
1.3.3 Protein phosphorylation ............................................................................................. .13
1.3.4 Regulated translation of localized mRNA s................................................................ 14
1.3.5 Local translation of mRNA shapes developmen t........................................................ 16
1.3.6 Local translation of mRNA in neuron .s...................................................................... 18
1.4 Quantification of gene expression and portein levels ........................................................ 20
1.4.1 Quantification of mRNA levels .................................................................................. 22
1.4.2 Quantification of protein levels .................................................................................. 23
1.4.3 mRNA levels as proxy for protein abundanc .e........................................................... .25
1.5 Fluorescence-based single cell resolution protein quantification ...................................... 26
1.5.1 Quantification of protein levels in single living cell .s................................................. 27
1.5.2 Protein production reporters must be carefully chose n............................................... 29
1.5.3 Approaches to visualizing locally translated protein .s................................................ 30
1.5.4 Requirements for a local protein synthesis reporte .r................................................... 34
1.5.5 Split GFPs are indicators of protein interactio n......................................................... .35
1.6 Figures ............................................................................................................................. 37
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1.7 Thesis introduction .............................................................................................................. 40
Chapter II - Quantification of mRNA and protein levels in single cells ................................ 41
2.1 Relation to overall thesis ..................................................................................................... 41
2.2 Introduction ......................................................................................................................... 42
2.3 Experimental design and detailed protocol ......................................................................... 44
2.3.1 Materials ....................................................................................................................... 45
2.3.2 Gene editing using CRISPR-Cas9 ................................................................................ 47
2.3.3 Single-cell protein level quantification ......................................................................... 49
2.3.4 Total RNA extraction ................................................................................................... 49
2.3.5 Reverse-transcription .................................................................................................... 50
2.3.6 Real-time polymerase chain reaction ........................................................................... 51
2.3.7 Calculation of absolute mRNA transcript number ....................................................... 52
2.3.8 Readout of amplification .............................................................................................. 53
2.3.9 Assay controls............................................................................................................... 54
2.3.10 Image acquisition and analysis ................................................................................... 55
2.4 Results ................................................................................................................................. 56
2.5 Discussion ........................................................................................................................... 59
2.6 Conclusion ........................................................................................................................... 62
2.7 Figures ................................................................................................................................. 64
Chapter III - A system for direct observation of subcellular protein translation in single
living cells. .................................................................................................................................... 79
3.1 Relation to overall project ................................................................................................... 79
3.2 Introduction ......................................................................................................................... 80
3.3 Materials and Methods ........................................................................................................ 83
3.3.1 Protein Quantification Reporter constructs .................................................................. 83
3.3.2 Split GFP DNA constructs ........................................................................................... 83
3.3.3 GFP1-10 protein production and extraction ................................................................. 84
3.3.4 GFP 11 peptides............................................................................................................ 85
3.3.5 Cell culture ................................................................................................................... 85
3.3.6 In vitro protein reconstitution ....................................................................................... 86
3.3.7 Endoplasmic reticulum and ribosome staining ............................................................. 86
3.3.8 Electrophysiology ......................................................................................................... 87
3.3.9 Image acquisition and analysis ..................................................................................... 87
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3.3.10 Statistical analysis....................................................................................................... 88
3.4 Results ................................................................................................................................. 89
3.4.1 GFP11 and GFP1-10 reconstitute spontaneously in vitro ............................................ 89
3.4.2 GFP reconstitution in vitro occurs at millisecond timescales ....................................... 90
3.4.3 GFP11 detects GFP1-10 in living cells ........................................................................ 91
3.4.4 GFP reconstitution can report sites of protein translation ............................................ 92
3.4.5 Proteins co-translated with GFP11 reporters function properly ................................... 94
3.4.6 GFP reconstitution can quantitatively readout protein translation ............................... 95
3.5 Discussion and conclusions ................................................................................................. 96
3.5.1 GFP1-10 fluorophore maturation ................................................................................. 97
3.6 Figures ............................................................................................................................... 100
Chapter IV - Applications and future directions of protein quantification using PQR ..... 109
4.1 Relevance to overall project .............................................................................................. 109
4.2 Applications of optical protein quantification using PQR ................................................ 110
4.2.1 Dynamic observation of protein synthesis in vivo ..................................................... 110
4.2.2 Optical normalization of protein production in vivo .................................................. 112
4.3 Split GFP as a quantitative marker of local protein synthesis in vivo .............................. 114
4.3.1 Generation of animals constitutively expressing GFP1-10 ............................................ 115
4.3.2 Local translation of Gurken protein in Drosophila oocytes ........................................... 117
4.3.3 Detection of local protein translation in living neurons ................................................. 121
4.4 Detection of local protein synthesis using PQR photoconvertible reporters. ................... 123
4.5 Figures ............................................................................................................................... 128
Chapter V - Thesis directions and conclusions ...................................................................... 137
References .................................................................................................................................. 142

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List of figures
Figure 1.1 Brightfield image of dissected whole ovarioles ......................................................... 37
Figure 1.2 PQR reporters allow quantification of protein production from cells. ....................... 39
Figure 2.1 Workflow of protein and mRNA measurement from the same cell. .......................... 64
Figure 2.2 Protein and mRNA measurement for multiple genes in a single cell. ........................ 66
Figure 2.3 Insertion of PQR-XFP reporters into the endogenous genomic loci of IgK and Rpl13a
using CRISPRs.............................................................................................................................. 68
Figure 2.4 Validation of CRISPR-mediated insertion of PQR-GFP in the endogenous IgK locus.
....................................................................................................................................................... 69
Figure 2.5 Illustration of the important steps and typical equipment used in the protocol. ......... 70
Figure 2.6 Titration of starting input cDNA volume. .................................................................. 72
Figure 2.7 Standard curve of serially diluted known amount of Rpl13a target. .......................... 73
Figure 2.8 Contamination of RNA sample quantification by genomic DNA can be assessed
using no-RT control reaction. ....................................................................................................... 74
Figure 2.9 Endogenous RNA and protein quantification from single cells. ................................ 75
Figure 2.10 Protein and mRNA relationships between multiple genes in single cells. ............... 76
Table 2.1 Sequences of primers and probes used in this protocol. .............................................. 78
Figure 3.1 Stoichiometric production of GFP11 reporters using PQR. ..................................... 100
Figure 3.2 In vitro characterization of the split GFP reconstitution reaction. ........................... 101
Figure 3.3 Reconstitution of split GFP occurs on the order of milliseconds in vitro. ............... 102
Figure 3.4 Split GFP reporters can be expressed using PQRs and the reconstitution of GFP
marks the presence GFP1-10 protein. ......................................................................................... 103
Figure 3.5 Split GFP reconstitution occurs at sites of active protein translation. ...................... 106
Figure 3.6 Co-translation of GFP11 reporters using PQR preserves the protein of interest’s
localization and function. ............................................................................................................ 107
Figure 4.1 PQR reporters are inserted in-frame into endogenous genes. .................................. 128
Figure 4.2 PQR constructs injected into mouse embryos result in red fluorescent pronuclei. .. 129
Figure 4.3 SplitGFP as a protein translation reporter. ............................................................... 131
Figure 4.4 GFP1-10 is expressed at high levels in transgenic animals. ..................................... 132
Figure 4.5 GFP11 can detect Gurken local translation in oocytes. ............................................ 134
Figure 4.6 Novel split fluorescent reporters exhibit more efficient reconstitution. ................... 136

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