The Role of Classical Nuclear Import Receptors during Myogenesis Open Access

Hall, Monica Nicole (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/fb494859t?locale=en
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Abstract


Abstract
The Role of Classical Nuclear Import Receptors during Myogenesis
By Monica Nicole Hall
Skeletal muscle is required for breathing, locomotion and metabolism. In adults,
myogenesis, the process of creating muscle is dependent on myogenic stem cells (satellite
cells) that have the ability to proliferate, differentiate and fuse into multinucleated cells
called myofibers in vivo or myotubes in vitro. Changes in gene expression during
particular stages of myogenesis are due to nuclear factors, such as myogenic transcription
factors, entering the nucleus and regulating gene expression. However, little is known
regarding the regulation of nuclear import of these nuclear factors in either mono or
multinucleated muscle cells. Classical nuclear import is one major pathway used by
eukaryotic cells to target proteins to the nucleus and is dependent upon a classical nuclear
localization signal (cNLS) within a protein that is recognized by the nuclear import
receptor family, karyopherin alpha (KPNA). Six KPNA paralogs exist in mouse:
KPNA1, KPNA2, KPNA3, KPNA4, KPNA6 and KPNA7. We established that five
KPNA paralogs are expressed by primary mouse myoblasts in vitro and that their steady-
state levels increase during differentiation. We used RNAi to identify paralog-specific
roles for KPNA1 and KPNA2 in muscle cell proliferation, while only KPNA2 had a role
in myotube growth. Furthermore, we determined that quiescent (non-proliferating)
satellite cells express four KPNA paralogs, Kpna2, Kpna3, Kpna4 and Kpna6, but not
Kpna1. Since Kpna1 is expressed in proliferating satellite cells, we investigated the role
of KPNA1 in satellite cell proliferation. Analysis of Kpna1+/- muscles revealed an
increase in satellite cell proliferation during muscle maintenance and regeneration
compared to Kpna1+/+ muscles. We hypothesize that KPNA1 may function in satellite
cells to import nuclear proteins required for the negative regulation of proliferation to
prevent over-proliferation and potential exhaustion of the satellite cell pool in skeletal
muscle. These results identify distinct import pathways that rely on specific KPNAs
suggesting that regulation of classical nuclear import has a critical role in controlling
gene expression during myogenesis. Uncovering the role of classical nuclear import in
regulating the nuclear import of proteins required for proper satellite cell function and
myotube growth could provide valuable insight into the critical role of nucleocytoplasmic
transport during myogenesis.


The Role of Classical Nuclear Import Receptors during Myogenesis
By
Monica Nicole Hall
B.S., University of Louisville, 2004
Advisor: Grace K. Pavlath, Ph.D.
A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in Genetics and Molecular Biology
Graduate Division of Biological and Biomedical Sciences
2011

Table of Contents




Table of Contents

Chapter 1: Background and Significance ........................................................................1

1.1 Skeletal Muscle ...........................................................................................................2

1.1.1 Skeletal Muscle Structure and Function ............................................................3

1.1.2 Adult Skeletal Muscle Origin, Model Systems and Morphology .......................4

1.1.3 Challenges of Studying Satellite Cell Function .................................................8

1.1.4 Gene Regulation during Myogenesis ...............................................................11
1.2 Nucleocytoplasmic Transport during Skeletal Myogenesis .....................................12

1.2.1 The Nuclear Envelope ......................................................................................12

1.2.2 Nuclear Pore Complexes .................................................................................14

1.2.3 Nuclear Import Pathways ................................................................................17
A. Classical Nuclear Import: Karyopherin Alpha Family ...............................18
B. Karyopherin Beta Family Members Mediate Import and Export ...............24
1.3 Identifying Classical Nuclear Import-Dependent Cargoes .......................................28
1.4 Remodeling of the Nuclear Transport Machinery ...................................................30
1.5 Challenges in Studying Nucleocytoplasmic Transport in Multinucleated Cells ......33
1.6 Summary ...................................................................................................................37
1.7 Figures.......................................................................................................................38
Chapter 2: Introduction ..................................................................................................48
Chapter 3: Materials and Methods ................................................................................52
Chapter 4: Distinct roles for Karyopherin Alpha Import Receptors during
Myogenesis ........................................................................................................................64
4.1 Introduction ...............................................................................................................65
4.2 Results .......................................................................................................................67
4.3 Discussion .................................................................................................................74
4.4 Figures.......................................................................................................................78
Chapter 5: The Role of Karyopherin Alpha1 in Satellite Cell Function ...................98
5.1 Introduction ...............................................................................................................99
5.2 Results .....................................................................................................................103
5.3 Discussion ...............................................................................................................110
5.4 Figures.....................................................................................................................115
Chapter 6: Discussion ....................................................................................................127
6.1 Introduction .............................................................................................................128
6.2 The Existence of Multiple Karyopherin Alpha Paralogs in Skeletal Muscle .........129
6.3 Classical Nuclear Import and Karyopherin Alphas Function in Satellite Cells......136
6.4 Identifying Classical Nuclear Import Cargoes during Myogenesis ........................139
6.5 Therapeutic Strategies .............................................................................................140
6.6 Summary .................................................................................................................142
6.7 Figures.....................................................................................................................144
References .......................................................................................................................150


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