Abstract
Microtubules are dynamic, filamentous polymers composed of
αβ-tubulin and are essential cytoskeletal components of
all eukaryotic cells. Microtubules are required for a variety of
essential cellular functions, including cell morphogenesis,
motility, mitosis, intracellular traffic, and formation of both
primary cilia and flagella. Highlighting their cellular importance,
microtubules are also the primary target for many cancer
therapeutics. In addition to post-translational modifications and
selective expression of tubulin isotypes, the functions of
microtubules are strongly regulated by the dynamics of the
individual microtubule polymers. Dynamic instability is a term that
describes the process by which microtubules stochastically
alternate between polymerization and depolymerization, which is
largely controlled by the binding and hydrolysis of tubulin-bound
guanine nucleotide. Despite our understanding of the many levels of
microtubule regulation, one fundamental aspect of microtubule
biology remains poorly understood: the biogenesis and assembly of
the αβ-tubulin heterodimer. Tubulin is composed of
α- and β-subunits, whose folding and assembly require a
set of tubulin-specific chaperones, or cofactors, that form a
series of folding intermediates in the eventual production of
native αβ-tubulin dimer. Described herein is a novel role
for the small, regulatory GTPase ARL2 in the cofactor-mediated
tubulin folding and heterodimer assembly pathway. ARL2 is shown to
strongly interact with the tubulin-specific chaperone D (TBCD),
forming a complex that I argue is integral to the tubulin folding
pathway. In contrast to previous models, I also propose that the
cycling of guanine nucleotides on ARL2 is a key regulatory
mechanism that is required for the folding and formation of native
αβ-tubulin heterodimers.
Table of Contents
CHAPTER 1: INTRODUCTION p.1
MICROTUBULES p.2
TUBULIN FOLDING p.4
Figure 1-1 p.5
TUBULIN-SPECIFIC CHAPERONE D p.7
ARF-LIKE 2 p.8
TBCS IN DISEASE p.12
FOCUS OF THIS DISSERTATION p.13
REFERENCES p.14
CHAPTER 2: A TRIMER CONSISTING OF THE TUBULIN-SPECIFIC CHAPERONE
TBCD, REGULATORY GTPASE ARL2, AND β-TUBULIN IS REQUIRED FOR
MAINTAINING THE MICROTUBULE NETWORK p.25
ABSTRACT p.26
INTRODUCTION p.26
RESULTS p.29
TBCD and ARL2 are present in animal cells and tissues in a complex
with an apparent molecular weight of ~200 kDa p.29
Figure 2-1 p.30
Figure 2-2 p.33
Recombinant GST-TBCD co-purifies with αβ-tubulin
p.34
Figure 2-3 p.36
Co-expression of GST-TBCD and ARL2 results in the formation and
ability to purify a TBCD/ARL2/β-tubulin trimer p.37
Figure 2-4 p.38
TBCE can bind to the TBCD/ARL2/β-tubulin trimer or to
TBCB/α-tubulin p.40
Figure 2-5 p.42
ARL2 mutants that lose TBCD binding have reduced ability to alter
microtubule densities p.44
Figure 2-6 p.46
Figure 2-7 p.48
DISCUSSION p.50
EXPERIMENTAL PROCEDURES p.57
REFERENCES p.64
CHAPTER 3: NUCLEOTIDE BINDING TO ARL2 IN THE
TBCD/ARL2/β-TUBULIN COMPLEX DRIVES CONFORMATIONAL CHANGES IN
β-TUBULIN p.69
ABSTRACT p.70
INTRODUCTION p.70
RESULTS p.75
The TBCD/ARL2/β-tubulin complex binds both GDP and GTP
p.75
Figure 3-1 p.77
Figure 3-2 p.79
Figure 3-3 p.81
Molecular dynamics of TBCD in the TBCD/ARL2/β-tubulin complex
are only minimally affected by the binding of guanine nucleotides
p.83
Figure 3-4 p.85
Addition of guanine nucleotides to the TBCD/ARL2/β-tubulin
complex results in conformational changes in β-tubulin that do
not map onto regions involved in guanine-nucleotide binding
p.86
Figure 3-5 p.87
HDX-MS analysis of ARL2 monomer reveals large changes, particularly
at consensus
GTP-binding motifs p.89
Figure 3-6 p.91
Formation of the TBCD/ARL2/β-tubulin complex results in
distinct changes in the molecular dynamics of ARL2 p.93
Figure 3-7 p.94
ARL2 binds guanine nucleotide in the TBCD/ARL2/β-tubulin
complex p.96
Figure 3-8 p.97
DISCUSSION p.99
EXPERIMENTAL PROCEDURES p.105
REFERENCES p.111
CHAPTER 4: DISCUSSION p.119
SUMMARY p.120
CHALLENGING THE STATUS QUO p.121
Figure 4-1 p.123
FUTURE DIRECTIONS p.126
Figure 4-2 p.127
CONCLUDING REMARKS p.129
REFERENCES p.131
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