Experimental projects for Macromolecules 2013.
Overview:
1. Molecular self-assembly: Using right angle light-scattering to follow oligomerization of IRE1
and kinetic analysis to understand the mechanism of its self-assembly.
2. Protein structure and interactions: Figuring out the structural determinants of sheet and
filament formation by a bacterial actin-like protein.
3. Membranes and mathematical modeling: Using quantitative models to understand the origin
and maintenance of multiple, membrane-bound compartments.
4. Mobility of membrane-associated proteins: Using single-molecule TIRF microscopy to study
the two-dimensional diffusion of proteins in different regions of the plasma membrane.
5. Assembly of complex cellular structures: Using the power of polarized light microscopy and
Stentor cell biology to follow the assembly of basal bodies and flagella.
6. Regulated protein-protein interactions: Regulating histone mark recognition by an HP1
protein
1. Molecular mechanism of IRE1 self-assembly.
Question: Does the stress-sensor, IRE1, assemble via a nucleation-condensation reaction? If so,
what is the size of the IRE1 nucleus? What are the rate constants for IRE1 polymer assembly?
Biological Context: Cells maintain quality control over secreted and transmembrane proteins in
the endoplasmic reticulum (ER), in part by using a biochemical pathway called the Unfolded
Protein Response (UPR). Accumulation of unfolded or partially folded proteins in the ER, which
can occur during development or disease, triggers the UPR and initiates a transcriptional
program that increases the folding
capacity of the ER. This is
accomplished by increasing the
production of chaperones and lipids
that expand the ER.
The best-studied UPR transducer is
Inositol Requiring Enzyme 1
(IRE1), an ER transmembrane
protein with kinase and
Figure 1. Structure of helical IRE1 filaments determined from x-ray
endoribonuclease (RNase) domains
crystallography (Korennykh et al., 2009).
in the cytoplasmic part of the
protein. Upon sensing ER stress, IRE1 oligomerizes (Figure 1) and undergoes transautophosphorylation to activate its non-conventional RNase activity. IRE1 specifically splices
the mRNA encoding XBP1 (or Hac1 in budding yeast). Spliced XBP1 mRNA is then translated
into a transcription factor that upregulates UPR-target genes.
Under ER stress conditions, IRE1 has been shown to form foci in vivo and to exhibit highly
cooperative splicing activity in vitro. Furthermore, IRE1 will reversibly self-assemble at high
protein concentration, forming a cloudy, heterogenous suspension that dissipates upon increasing
salt concentration. As reflected by its in vivo activity, oligomer formation of IRE1 is “switchlike,” making it difficult to identify the minimum number of monomers required for activity. To
better understand the cooperative activation of IRE1 and to provide deeper insight into ER stress
responses, we will use light scattering to follow the kinetics of IRE1 self-assembly. We will then
use mathematical tools developed for studying assembly of cytoskeletal filaments to characterize
the assembly mechanism of IRE1 polymers.
Tools:
- Purified IRE1 protein (wildtype and drug-sensitized mutant).
- ATP-competitive inhibitor (1NM-PP1) that induces polymerization.
- Stopped-flow rapid mixer
- Quartz Cuvettes
- Fluorometer
Battle plan:
1) Use steady-state, right-angle light scattering to determine the critical concentration for selfassembly of IRE1.
2) Use time-resolved light scattering to follow the kinetics of IRE1 assembly.
3) Perform two types of analysis (Nishida and Sakai / Flyvbjerg and Leibler) to determine the
size of the IRE1 nucleus.
4) Build a kinetic model for IRE1 assembly in Kinsim or Matlab and fit it to experimental data.
Background Papers:
Korennykh A, et al. (2009). The unfolded protein response signals through high-order assembly
of Ire1. Nature 457:687-693.
Papa F.R., et al. (2003). Bypassing a Kinase Activity with an ATP-competitive Drug. Science
28:1533-1537.
Flyvbjerg H, Jobs E, Leibler S. (1996) Kinetics of self-assembling microtubules: an "inverse
problem" in biochemistry. Proc Natl Acad Sci U S A. 93(12):5975-9.
Polka JK, Kollman JM, Agard DA, Mullins RD. (2009) The structure and assembly dynamics of
plasmid actin AlfA imply a novel mechanism of DNA segregation. J. Bacteriol. 191(20):
6219-6230.
2. Atomic structure and polymer architecture of a bacterial actin-like protein.
Question: What are the structural features that determine the architecture of bacterial
cytoskeletal polymers?
Biological Context: Alp7A is an actin-like protein (Alp) found on large, low-copy plasmids in
Bacillus species. It assembles into filaments that segregate plasmids and ensure that each
daughter cell inherits at least one plasmid following cell division. Conventional actin filaments
and the best-studied bacterial Alps (e.g. ParM) form polarized, two-stranded filaments. In vitro
Alp7A, however, forms twisted sheets or ribbons of two-stranded filaments, stacked side-by-side
in anti-parallel rows. This sheet is not likely to be the DNA-segregating form of the polymer but
the anti-parallel arrangement of filaments in the sheet probably reflects a protein-protein
interaction required for Alp7A function. Anti-parallel association would enable Alp7A filaments
Figure 1. Left: Crystal structures of actin, Alp7A, and ParM. The massive C-terminal helix of Alp7A is
marked by a red arrow. Right: Electron micrograph of Alp7A polymer sheets. Inset: Averaged images of
Alp7A sheets showing anti-parallel arrangement of filaments
to form a bi-polar, force-generating structure, analogous to a mitotic spindle, that pushes
plasmids in opposite directions.
We do not understand the structural basis of anti-parallel filament stacking but the atomic
structure of Alp7A reveals some unique features that might help explain it. The most unique
feature of the Alp7A structure is a long, straight alpha-helix at the very C-terminus of the protein.
This helical region is much longer than in any other previously studied actin-like protein. To
determine whether this helical region mediates lateral interactions we have made a series of
truncation mutants. These have not been previously purified and we will use them to determine
whether the C-terminal tail contributes to Alp7A filament structure and/or dynamics.
Tools and Techniques:
- Wildtype and truncation mutants of Alp7A.
- Uranyl-formate negative stain
- Electron microscope
- Flurometer to monitor right-angle light scattering
Battle Plan:
1. Purify Wildtype and mutant Alp7A proteins.
2. Induce polymerization with millimolar concentrations of ATP
3. Determine the ultrastructure of Alp7A filaments by electron microscopy of negatively stained
samples.
4. Use right-angle light scattering to follow the kinetics of Alp7A polymerization.
Background Papers:
Garner EC, Campbell CS, Weibel DB, Mullins RD. (2007) Reconstitution of DNA segregation
driven by assembly of a prokaryotic actin homolog. Science. 315(5816):1270-4.
Gayathri P, Fujii T, Møller-Jensen J, van den Ent F, Namba K, and Löwe J (2012). A Bipolar
Spindle of Antiparallel ParM Filaments Drives Bacterial Plasmid Segregation. Science.
Published online 25 October 2012
Derman AI, Becker EC, Truong BD, Fujioka A, Tucey TM, Erb ML, Patterson PC, Pogliano J.
(2009) Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in
bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A. Mol
Microbiol. 73(4):534-52.
Polka JK, Kollman JM, Agard DA, Mullins RD. (2009) The structure and assembly dynamics of
plasmid actin AlfA imply a novel mechanism of DNA segregation. J. Bacteriol. 191(20):
6219-6230.
3. Modeling membrane movement and the establishment of distinct cellular compartments
Question: Can simple kinetic and thermodynamic rules explain the origin and stability of
distinct cellular compartments?
Biological context: All eukaryotic cells use vesicular trafficking to transport proteins and lipids
(Barlowe, 2000). Vesicles bud from one compartment, taking along both soluble and membrane
proteins as well as lipids, and fuse with another compartment. Transport in the anterograde
direction must be counterbalanced by retrograde traffic to keep the size of compartments
constant and reuse components of the transport machinery. The bidirectional traffic would tend to
equalize the composition of the compartments, yet most proteins and some lipids are
concentrated in one organelle and define its identity. How can such nonuniform distributions be
achieved?
The classic explanations for the generation and stability of distinct compartments fail to explain
the ultimate origins of asymmetry. Often these explanations simply shuffle the big question
around like an ace of spades in a crooked game of three-card monty. For example, t-SNAREs
have been suggested to determine the identity of a compartment by directing fusion of specific
vesicles, but how are the SNAREs themselves localized? SNAREs may be concentrated
according to the cholesterol content of a membrane, but, in this case, how would cholesterol be
localized? The same kind of circularity arises if one assumes the specific binding of a coat to a
compartment. How does a coat “know” where to go? The specific coat binding would have to be
caused by the local- ization of certain proteins, and again the question is how these are targeted.
Thus, despite enormous insight into the molecular details of vesicular trafficking, the
fundamental problem of how compartments are generated is still unanswered. Moreover, an
explanation is needed why a nonhomogeneous distri- bution of protein and lipid is a stable state.
The robustness of the system is best illustrated by experiments with the drug brefeldin A (BFA):
when added to cells, the Golgi enzymes are redistributed back into the ER, but when the drug is
removed, they return to their original position (Lippincott-Schwartz et al., 1990). The experiment
also raises the question of why the Golgi grows back to its original size. How is the size of an organelle generally determined?
In this project, you will test a mathematical model proposed by Heinrich and Rapoport (2005).
The model is based on a minimal vesicular transport system, with coats and SNAREs as basic
components. Can this simple system generate stable, nonidentical compartments? Can it explain
not only the origin of specific compartments, but also their size regulation and the transport of
cargo proteins to different cellular destinations? How does the model compare to previous
attempts to explain asymmetry (Glick et al., 1997; Weiss and Nilsson, 2000)? Can you propose a
different, more plausible, or more successful model?
Tools:
- Matlab
- Berkeley Madonna
- The human brain
Battle plan:
1. Download code from Rapoport laboratory and recapitulate results.
2. Identify the essential features of the Rapoport model and try to identify a simpler system that
recaptures the behavior of the original model.
3. Write a stochastic simulation of vesicle traffic to see whether the mechanism still works when
the number of vesicles is VERY small.
4. Propose and test a competing model for establishment of distinct compartments.
Background papers:
Membranes
Heinrich R, Rapoport TA. (2005) Generation of nonidentical compartments in vesicular transport
systems. J Cell Biol. 168(2):271-80.
Barlowe C. (2000) Traffic COPs of the early secretory pathway. Traffic. 1(5):371-7. Review.
Modeling
Vilar JM, Guet CC, Leibler S. (2003) Modeling network dynamics: the lac operon, a case study.
J Cell Biol. 161(3):471-6. Review.
http://dephnis.tic.unam.mx/cursos/complex/biblio/
Strogatz1994NonlinearDynamicsandChaos.pdf
4. Mobility of membrane proteins
Question: We know that many things can affect protein mobility, including confinement and
interaction with binding partners. Here, we will ask: how does the mobility of a membrane
protein (G-protein Coupled Receptor) change inside and outside of clathin coated pits?
Biological Context:
Cells constantly sample their environment and adapt accordingly in order to survive, whether
they are unicellular or part of a larger organism. The most common method of sampling is via
transmembrane receptors in the plasma membrane, which undergo conformational changes upon
ligand binding and induce signaling within the cell. Many of these receptors (such as the model
beta-2 adrenergic receptor) are then internalized to the endosome via clathrin dependent
endocytosis, and can be differentially sorted and either recycled back to the PM or degraded at
the lysosome / Golgi.
How a receptor ends up in a clathrin coated pit is not well understood, although many posttranslational modifications have been identified that regulate this process, including
phosphorylation and/or ubiquitination of the receptor’s cytoplasmic tail. Several models for the
movement of a receptor into a coated pit have been proposed, although none have been
decisively tested. The emergence of super resolution microscopy and single particle tracking
techniques enable us to tackle the question of how an activated GPCR associates with clathrin
coated pits.
Tools/Techniques:
-Cell lines: HEK293, ssB2AR (stable cell line)
-Constructs: B2AR-Flag, B2AR-mEOS, µOR-mEOS, D1-mEOS
-SPIT-PALM super resolution microscopy, TIRF Microscopy
-STORM, TIRF microscopes in the Nikon Imaging Center and the Mullins Lab
-Matlab, ImageJ
Battle Plan:
1. TIRF imaging of bulk flow of the Beta-2 adrenergic receptor (B2AR) into clathrin coated pits
(CCP) upon ligand-induced activation.
2. Super resolution SPIT-PALM imaging of B2AR-mEOS; single particle tracking into CCP
upon ligand-induced activation.
3. Computational modeling of the movement of the receptors into CCP and comparison with the
data collected.
4. Targeted disruption of the system (actin?) to probe the mechanism of receptor capture in CCP.
5. Compare with similar experiments using different receptors that do not aggregate well in CCP.
Background Papers:
Diffusion to Capture Lecture
http://www.chem.utah.edu/ibac/pdf/Week%206%20Part%201%20Ryan%20White.pdf
SptPALM
Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J.
(2008) High-density mapping of single-molecule trajectories with photoactivated localization
microscopy. Nat Methods. 5(2):155-157.
Beta Receptor Clustering in Clathrin Coated Pits
Puthenveedu MA, von Zastrow M. (2006) Cargo regulates clathrin-coated pit dynamics. Cell.
127(1):113-124.
5. Using the power of polarized light microscopy and Stentor cell biology to follow the
assembly of basal bodies and cortical microtubules.
Question: Can we use polarized light microscopy to figure out how Stentor coeruleus creates
new basal bodies and new cortical rows?
Biological Context: Centrioles play important roles in Eukaryotes. They organize the
microtubule cytoskeleton during interphase and the mitotic spindle during mitosis as part of their
function in the centrosome. Additionally, as basal bodies they act as templates for cilia and
flagella. Despite years of work we know little about how cells assemble centrioles. Stentor
coeruleus is an exciting new model in which we can address questions of centriole assembly.
Stentor is a unicellular organism that is 1mm long and possesses a highly polarized cell cortex.
The cell geometry is defined by cortical rows composed of microtubule bundles, and these
bundles of microtubules are organized by basal bodies that produce cilia along the length of the
organism.
Although Stentor lacks a centrosome, individual cells have tens of thousands of basal bodies and
could be a powerful tool for studying centriologenesis but to date there have been no studies of
this in Stentor and so the techniques have not been developed. One basic question that needs to
be addressed is where do cells initiate the assembly of new centrioles? Unfortunately many of
the modern tools that would allow us to directly answer these questions, such as the GFP-tagging
of centriolar proteins, have not yet been adapted for use in Stentor and so alternative approaches
need to be taken. One potentially powerful tool is polarized light microscopy and the LC-pol
scope. We could potentially use polarized light microscopy to visualize centrioles and their
associated structures, which are all composed of microtubules, in living cells.
In this project you will use polarized light microscopy as a tool to visualize the formation of new
basal bodies in living Stentor. Despite their abundance in Stentor, little is known about how,
where, and when the cell undergoes the assembly of new centrioles. Do they constantly form
throughout the cell cycle to account for cell growth or is there a burst of production before
division? Is this growth limited to one region of the cell or does it occur globally? Are there
patterns, or waves, of centriologenesis or is production uniform?
Tools/Techniques:
LC-Pol scope
Stentor coeruleus handling
Antibodies against centrin for immunofluorescence
Battle Plan:
1. Immunofluorescence of centrin (obtain a baseline for the density of centrioles in the rows)
2. Time lapse imaging of Stentor growth (will require paralyzing the cells with NiCl2)
3. LC-pol scope imaging of basal bodies and their associated microtubule bundles in Stentor. Use
birefringence to count the microtubules in the cortical rows.
4. Time lapse imaging of on the Stentor LC-pol scope.
Background Papers:
Pearson C.G. and Winey M., (2009) Basal body assembly in ciliates: The power of numbers.
Traffic 10: 461-471. Review
Huang B. and Pitelka D.R., (1973) The contractile process in the ciliate, Stentor coeruleus. J Cell
Biol. 57, 704-728. (Best EM of cortical structures, contractile process itself is not all that
important)
Oldenbourg R, Salmon ED, Tran PT. (1998) Birefringence of single and bundled microtubules.
Biophys J. 74(1):645-54.
Inoue S. (1981) Cell division and the mitotic spindle. J Cell Biol. 91(3 Pt 2):131s-147s. Review.
6. Regulating histone mark recognition by an HP1 protein
Question: How does phosphorylation and a putative oligomerization interface regulate histone
mark recognition by the heterochromatin protein HP1?
Biological context: Large stretches of the genome are repressed by formation of a set of
structures, collectively called heterochromatin. These structures involve the formation of a
complex between the protein HP1 and chromatin that is methylated on histone H3 at lysine 9
(H3K9). It has been hypothesized that HP1 proteins can cooperatively spread across chromatin
and promote chromatin condensation. At a molecular level, how this oligomerization occurs is
not fully understood. It is also not clear how the assembly and disassembly of HP1 proteins is
regulated. All HP1 proteins have a chromodomain (CD) that recognizes the H3K9methyl mark, a
chromoshadow domain (CSD) that forms a head to head dimer and a hinge region that interacts
with nucleic acids (Fig.1). The fission yeast HP1 protein, Swi6, contains a loop in its CD that
appears to perform three different types of functions depending on context: (i) in the absence of
methylated nucleosomes, the loop of one CD occupies the histone tail binding of another CD
within a dimer resulting in an auto-inhibited state; (ii) in the presence of a methylated
nucleosome, the loop is flipped out by a histone tail and the flipped out loop participates in
binding nucleosomal DNA: (iii) in the context of nucleosome arrays, the flipped out loop forms
an oligomerization interface between dimers (Fig.1). Swi6 also contains a stretch of glutamates
in the N-terminal region of the CD, which are important for binding the histone H3 tail.
Human HP1shows a few
interesting differences (Fig.1):
(i) it does not have the same
loop sequence as Swi6 and so
it is not known if the loop has
the same role as in Swi6; (ii)
hHP1 has serines at its Nterminus instead of the stretch
of glutamates found in Swi6
and phosphorylation of these
serines increase
heterochromatin formation in
vivo and H3 tail binding in
vitro. To characterize how the
different features of HP1
cooperate to recognize the
methylated H3 tail, we will
measure the binding affinities
of a series of HP1 mutants for
the methylated H3 tail using fluorescence anisotropy. We will then interpret the results in terms
of how the different domains in HP1 might cooperate to recognize the methylated H3 tail.
Tools:
-Purified WT HP1, Phosphorylated HP1, phosphorylated and un-phosphorylated HP1 loop
mutants, CSD mutant HP1
- Fluorescein labeled H3K9me3 peptide
-96 well plates
-Fluorimeter
Battle plan:
1) Determine how the fluorescence anisotropy of the H3 peptide changes as a function of HP1
concentration.
2) Fit the data using different models for HP1-peptide binding
3) Compare the Kd values obtained for all the mutants to determine how the different domains of
HP1 contribute to recognizing the methyl mark and how phosphorylation regulates the affinity.
Background papers:
1. Canzio D et al, (2013) A conformational switch in HP1 releases auto-inhibition to drive
heterochromatin assembly. Nature; 496:377-81.
2. Canzio D et al, (2011) Chromodomain-mediated oligomerization of HP1 suggests a
nucleosome-bridging mechanism for heterochromatin assembly. Mol Cell. 41:67-81
3. Hiragami-Hamada K et al. (2011) N-terminal phosphorylation of HP1{alpha} promotes its
chromatin binding. Mol Cell Biol. 31:1186-200.