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Analyzing Spatial Point Patterns in Biology
Dr. Maria Byrne
Mathbiology and Statistics Seminar
September 25th, 2009
Analyzing Spatial Point Patterns in
Membrane Biology
Dr. Maria Byrne
Mathbiology and Statistics Seminar
September 25th, 2009
OutlineA. Biomembranes
B. lipid organization?
C. Statistical Analysis of Point Patterns- Ripley’s K
D. Conclusions and Questions
Overview
The micro-organization of lipids and proteins within the cell membrane is an important but open question.
We investigate ways in which statistical methods could be used to determine existence and properties of lipid organization in cell membranes.
A. Biomembranes
Biological Membranes
Structurally composed of phospholipids. Phospholipid: hydrophobic part and hydrophilic part
Naturally form bilayers.
Biological Membranes
Structurally composed of phospholipids. Phospholipid: hydrophobic part and hydrophilic part
www.bioteach.ubc.ca
Biological Membranes
Structurally composed of phospholipids. Phospholipid: hydrophobic part and hydrophilic part
http://academic.brooklyn.cuny.edu/biology
Sea of phospholipids, with a diverse variety of proteins and lipids.
B. Lipid Organization
Are lipids and proteins arranged randomly
throughout the biomembrane, or is there micro-organization?
Hee
tder
ks a
nd W
eiss
Lipid-Lipid Interactions
Gel Domains: Phospholipids with long, ordered chains
Fluid Domains: Phospholipids with short, disordered chains
Cholesterol : Gel domains form a liquid ordered phase
Domain Formation In Model Membranes
The Lipid Raft Hypothesis The cell membrane phase separates into liquid-ordered domains and liquid-disordered domains.
Liquid-Ordered Domains
- “lipid rafts”
- enriched in glycosphingolipids and cholesterol
- act to compartmentalize membrane proteins: involved in signal transduction, protein sorting and membrane transport.
Applications/Relevance
Immune system: Lipid domains are putatively required for antigen recognition (and antibody production).
Vascular system: lipid domains are putatively required for platelet aggregation.
HIV: lipid domains are putatively required to produce virulogical synapses between T-lymphocytes that ennable replication
Cancer: Ras proteins, implicated in 30% of cancers, are thought to signal by compartmentalizing within different domains
Hypotheses for lipid organization:
• Random / homogeneous distributions• Complexes/Oligomers• Exotic organizations
Investigate how to quantitatively distinguish (a) from (b) below:
Future Directions
Investigate how to quantitatively distinguish (a) from (b) below:
Future Directions
Investigate how to quantitatively distinguish (a) from (b) below:
Investigate other models for domain formation:
Oligomerization (e.g., mass action)Cell-controlled organization Protein “corals”
Lipid Raft Controversy
• Lipid rafts: Elusive or Illusive? (S. Munro, Cell, 2003)• Recent controversy surrounding lipid rafts. (M. Skwarek, Arch Immunol Ther Exp ,
2004)– “Although there have been many articles concerning LRs, there is still controversy about
their existence in the natural state, their size, definition, and function.” • Lipid Rafts: Real or Artifact? (M. Ediden, Science Signaling Opening Statement,
2001)• Lipid rafts: contentious only from simplistic standpoints (J. Hancock, Opinion in
Nature Reviews Mol Cell Bio, 2004)• Lipid Rafts Exist as Stable Cholesterol-independent Microdomains in the Brush
Border Membrane of Enterocytes (Hansen et al, Journal of Biol Chem, 2001)• Special Issue: Lipids Rafts (BBA, 2005)
– The controversy arises from the fact that rafts have proven frustratingly difficult to precisely define in cells. We do not yet have an unambiguous picture of raft size, stability, or protein and lipid composition. It is also not clear whether rafts exist in cell membranes constitutively or form only in a regulated manner.
Adding to complexity/lack of consensus:
• Many cell types
• Different types of cell membranesmembranes of organellesplasma membrane
• Inner verses outer membrane layers
C. Statistical Analysis of Point Patterns
•The positions of N molecules is precisely described by 2N numbers in continuous space.
Prior, Muncke,Parton and Hancock
Yet…
The organization of lipids are determined by physical and biological parameters that may greatly constrain the set of possible distributions.
Example: if the distribution of lipids is genuinely random, the entire distribution can be described with just the lipid density.
Ripley’s K
Aggregation and Segregation
Aggregation and Segregation
Domain Radius
• A positive value of H(r) indicates clustering over that spatial scale.
• A negative value of H(r) indicated dispersion over that spatial scale.
• The maximum value of H(r) occurs at r = the radius of maximal aggregation.
• To what extent does this maximum report on the domain radius?
Domain Radius
• To what extent does this maximum report on the domain radius?
Method used in • R.G. Parton et al., J. Cell Biol., 2004
• Hancock and Prior, Trends Cell Biol, 2004
• Zhang et al., Micron, 2006
Domain Radius To what extent does this maximum report on
the domain radius?
Prediction correct within a factor of 2, but the radius is usually over-estimated.
Domain Radius Prediction correct within a factor of 2, but the
radius is usually over-estimated.
Why: the radius of maximal aggregation occurs when the effects of aggregation within a domain begin to be offset by the dispersion outside the domain, which occurs at a radius somewhere beyond the domain boundary.
Domain Radius Solution: remove accumulative effects by taking the derivative of
H(r).
Instead of identifying the domain radius, we identify the domain diameter by finding the value of r when the derivative is minimized.
Domain Radius
Application: K-ras Nanoclusters
Experimentally derived point pattern with an immunogold density 625 m-2.
Application: K-ras Nanoclusters
Nanoclusters of constant size (~16nm).
Each contains ~3.2 gold particles.
Noise: 56% protein monomeric
Application: K-ras Nanoclusters
Method:
(1) Use Ripley’s K to estimate domain size for the experimental image. (14 nm)
(2) Use Monte Carlo generated images to estimate error due to noise.
Effect of “Non-Random” Noise
•Points non-randomly dispersed within domains.(significant effect!)
•Domains not approximately disk-shaped.(significant effect!)
•Interconnected or finger-like domains.(method: useless)
Immediate challenge: how to ‘parametrize’ non-random noise?
D. ConclusionsAnd Questions
Conclusions• Ripley’s K can be used to determine
domain radius if points are arranged randomly within domain.
• The measure is robust to random noise.
• The measure is sensitive to systematic / patterned noise.
Question• It’s relatively straight-forward to measure the extent
to which a pattern holds.
• Determining which patterns hold is an ad hoc process.– Are the particles aggregated?– Are the particles separated?– Are the particles arranged in predetermined ways?
• More difficult to know if any pattern holds.
• To what extent can the problem of “finding pattern” be framed so that it may be (more) well-defined? In general, how can one identify pattern?
A Random Donor is Excited
FRET Efficiency is Related to the Distance of the Nearest Acceptor
Compare with the Hausdorff Measure
The Hausdorff distance is the longest distance you can be forced to travel by an adversary who chooses a point in one of the two sets, from where you then must travel to the other set.
Compare with the Hausdorff Measure
The Hausdorff distance is the longest distance you can be forced to travel by an adversary who chooses a point in one of the two sets, from where you then must travel to the other set.
Compare with the Hausdorff Measure
),( YXd H
),(infsup),,(infsupmax yxdyxd
XxYyYyXx
Compare with the Hausdorff Measure
Compare with the Hausdorff Measure
The Hausdorff metric will measure one half the domain separation as long as the domain separation is greater than the domain radius.
Let X={inter-domain space} Y={domain space}