Bioelectric Processes of
Cellular Plasticity and Regeneration
Cellular Reprogramming Laboratory
Journal Club, April 6th, 2012
Bradly Alicea
http://www.msu.edu/~aliceabr/
Papers from the Tufts Regenerative
and Developmental Biology group
Michael Levin, PI
Role of Membrane Potential in the
Regulation of Cell Proliferation and
Differentiation. Stem Cell Reviews and
Reports, 5, 231-246 (2009).
Sundelacruz, Levin, and Kaplan
Bioelectric mechanisms in regeneration:
unique aspects and future perspectives.
Seminars in Cell and Developmental
Biology, 20, 543-556 (2009).
Levin
Bioelectrical Activity
Bioelectrical activity:
* used in some bony and cartilagenous
fishes for navigation, prey detection, and
communication.
* used in vertebrates and invertebrates for
driving muscle contraction, used in
communication and movement.
Bioelectrical Activity
Bioelectrical activity:
* used in some bony and cartilagenous
fishes for navigation, prey detection, and
communication.
* used in vertebrates and invertebrates for
driving muscle contraction, used in
communication and movement.
* ion channels used to specify functional
effect in a cell (e.g. tetanic stimulation,
LTP, muscle contractions).
* mode of transmission most important
aspect of effects (e.g. electrical field,
diffusion, flux along a gradient).
Wanted: a membrane potential
Membrane Potential vs. Electrical Fields
Cellular bioelectric potential expressed in terms of
dipoles:
Electrical Field (dipole with no
intermediate barrier).
Membrane potential (dipole with
selective permeability through a
barrier).
Membrane Potential vs. Electrical Fields
Cellular bioelectric potential expressed in terms of
dipoles:
Electrical Field (dipole with no
intermediate barrier).
Vmem
Membrane potential (dipole with
selective permeability through a
barrier).
Ion concentrations form
gradients between inside
and outside of membrane
via ion channels.
Fluxes vs. Gradients
Fluxes: changes in the flow of ions over time.
* channels open and close – introduces
selective permeability.
* “bursty” response functions (e.g. action
potentials).
Fluxes vs. Gradients
Fluxes: changes in the flow of ions over time.
* channels open and close – introduces
selective permeability.
* “bursty” response functions (e.g. action
potentials).
Gradients: difference in concentration of ions
over space.
* difference between inside and outside of cell
membrane – depolarized state leads to cell
excitation.
Consequence: release of neurotransmitters,
regulation of mRNA pools, etc.
Transport of Electrical Signals
Sundelacruz, Levin, and Kaplan paper, Table 1
Multiple mechanisms for transducing electrical signals:
* conformation changes in membrane proteins, electroosmosis.
* voltage-sensitive small-molecule transporters, translocation.
* electrophoresis of morphogens, redistribution of changed receptors in cell surface.
Do non-excitable cells have ion
channels? Yes!
He et.al FEBS Letters, 576(1-2), 156-160 (2004):
* cardiac fibroblast proliferation is mediated through ion channel activity (3
heterogeneously-expressed channel types).
Ca2+-activated K+ current - BKCa
Block, reduced proliferation
Volume-sensitive chloride current - I(Cl.vol)
Block, reduced proliferation
Voltage-gated sodium (INa)
Block, no effect
Do non-excitable cells have ion
channels? Yes!
He et.al FEBS Letters, 576(1-2), 156-160 (2004):
* cardiac fibroblast proliferation is mediated through ion channel activity (3
heterogeneously-expressed channel types).
Ca2+-activated K+ current - BKCa
Block, reduced proliferation
Volume-sensitive chloride current - I(Cl.vol)
Block, reduced proliferation
Voltage-gated sodium (INa)
Block, no effect
Main effects of induced channel dysfunction on proliferation (using pharmacology,
siRNA):
* accumulation of G0/G1 phase cells.
* after use of channel blockers, a reduced number of cells in S-phase.
* decrease in expression of Cyclin D1, E (cell cycle related genes).
Membrane Potential, Cellular
Functions
Models for ionic dysregulation and proliferation/cell cycle progression (Sundelacruz,
Levin, Kaplan paper):
MCF-7 breast cancer model: hyperpolarization of K+ channels = cell cycle
regulation.
* requires Vmem hyperpolarization during G0/G1 transition. K+ channel inhibition =
accumulation of cyclin-dependent p21 (blocks G1/S transition).
Membrane Potential, Cellular
Functions
Models for ionic dysregulation and proliferation/cell cycle progression (Sundelacruz,
Levin, Kaplan paper):
MCF-7 breast cancer model: hyperpolarization of K+ channels = cell cycle
regulation.
* requires Vmem hyperpolarization during G0/G1 transition. K+ channel inhibition =
accumulation of cyclin-dependent p21 (blocks G1/S transition).
Example of membrane polarization from neurons:
Depolarization:
Positive-going or positive
membrane potential.
Hyperpolarization:
Negative-going or negative
membrane potential.
COURTESY:
http://bioserv.fiu.edu/~walterm/GenBio2004/
* inhibits rise of action
potential.
Membrane Potential, Cellular
Functions
1) Model of hEAG (human ether a go go) activity
during cell cycle (breast cancer cells):
* activated during early G1 phase, Vmem depolarized
to -20mV.
* as hEAG upregulated during late G1, Vmem
hyperpolarization and Ca2+ entry.
COURTESY:
http://wwwsciencephoto.com
Codes
for
protein Kv11.1:
cardiac rhythm
and
cancer
establisher.
* further hyperpolarization drives G1/S transition.
2) Glioma model: inwardly-rectifying Kir4.1 channel
during proliferation:
* expressed
astrocytes.
specifically
in
glial-differentiated
COURTESY:
Figure 7,
Neuroscience,
129(4), 1043–
1054.
Measuring Membrane Potential
Vmem levels correlated with mitosis, DNA synthesis, cell cycle progression.
* resting potential corresponds with proliferative potential.
* somatic cells are hyperpolarized, tend to be quiescent, do not undergo mitosis.
Vmem: measured by dye imaging and electrophysiology.
Measuring Membrane Potential
Vmem levels correlated with mitosis, DNA synthesis, cell cycle progression.
* resting potential corresponds with proliferative potential.
* somatic cells are hyperpolarized, tend to be quiescent, do not undergo mitosis.
Vmem: measured by dye imaging and electrophysiology.
Dye Imaging (e.g. FRET)
COURTESY: Pacific Northwest National Labs
Measuring Membrane Potential
Vmem levels correlated with mitosis, DNA synthesis, cell cycle progression.
* resting potential corresponds with proliferative potential.
* somatic cells are hyperpolarized, tend to be quiescent, do not undergo mitosis.
Vmem: measured by dye imaging and electrophysiology.
Dye
Dye Imaging
Imaging (e.g.
(e.g. FRET)
FRET)
COURTESY: Pacific Northwest National Labs
Electrophysiology
Voltage-dependent Plasticity
Examples of Voltage-dependent
Plasticity
Spontaneous proliferation: accompanied by Vmem depolarization.
* K+ currents support proliferation and cell cycle progression.
* K+ flux resulting in depolarization favor proliferation.
Examples of Voltage-dependent
Plasticity
Spontaneous proliferation: accompanied by Vmem depolarization.
* K+ currents support proliferation and cell cycle progression.
* K+ flux resulting in depolarization favor proliferation.
Astrocytes: cells endogenously switch from quiescent to proliferative state
(triggered by response to injury).
* only some astrocytes (depolarized resting Vmem and specialized K+ channels)
will respond to injury.
Examples of Voltage-dependent
Plasticity
Spontaneous proliferation: accompanied by Vmem depolarization.
* K+ currents support proliferation and cell cycle progression.
* K+ flux resulting in depolarization favor proliferation.
Astrocytes: cells endogenously switch from quiescent to proliferative state
(triggered by response to injury).
* only some astrocytes (depolarized resting Vmem and specialized K+ channels)
will respond to injury.
Vascular smooth muscle cells: phenotypic switching due to injury.
* changes in ion channel composition (many different types involved).
How injured tissues “break the
membrane barrier”
In cases of injury, cell membrane is
disrupted:
A) positively-charged ions quickly
penetrate inside of the cell (NOT
through conventional means).
B) disruption creates an expedient
dipole, hence a locally strong current.
C) creates a current by which trophic
signals can be guided to the site of
injury.
Levin Review, Figure 2
Functional Phenotypes Enforced by
Electrophysiology
Lauritzen, I., et.al. K+-dependent cerebellar granule neuron apoptosis. Role of
task leak K+ channels. Journal of Biological Chemistry, 278, 32068–32076
(2003).
* K+-dependent developmental apoptosis (HC, Cerebellum).
COURTESY: Nature Reviews Molecular
Cell Biology 5, 614-625 (2004)
Functional Phenotypes Enforced by
Electrophysiology
Lauritzen, I., et.al. K+-dependent cerebellar granule neuron apoptosis. Role of
task leak K+ channels. Journal of Biological Chemistry, 278, 32068–32076
(2003).
* K+-dependent developmental apoptosis (HC, Cerebellum).
* K+ channel subunits = 4
TM, 2 P domains.
* TASK 2p phenotype leaky
when closed (mutant).
* expression = death in
proper conditions (e.g. pH).
COURTESY: Nature Reviews Molecular
Cell Biology 5, 614-625 (2004)
* important in 1:1 granularPurkinje cell matching.
Electrophysiology as Trigger of a
Cascade?
Does electrophysiology give us
complementary information to
biochemistry and other cellular
processes?
Yes!
Sundelacruz, Levin, and Kaplan review, Figure 1
Electrophysiology as Trigger of a
Cascade?
Does electrophysiology give us
complementary information to
biochemistry and other cellular
processes?
Yes!
* affects a physiological process
through proximal transduction
mechanism.
* amplified by secondary responses
and transcriptional effectors.
* results in a cellular “behavior”.
Sundelacruz, Levin, and Kaplan review, Figure 1
* aggregate cellular behavior gives
us the type and number of each cell
type.
Specific Functionality of the
Morphogenetic Field
Formula for a Morphogenetic Field
Morphogenetic field:
* growth, regeneration during
development, aging, and injury.
Is an additive combination of:
* bioelectric effects (ion channel
activity).
* biomechanics (tension, forces).
* extra-cellular
dynamics.
matrix
*
chemical
(microenvironment).
Michael Levin’s “formula” for development and
regeneration (Levin review, Figure 1)
(ECM)
effects
Signaling has a multiplicative
effect (combinatorial).
Special Function #1: Morphogenesis
Cell “coupled” through electrical signals – gap junctions.
1) Organize cells into functional domains.
* delimit populations of neuronal cells during spinal cord development.
1
2
J. Cell Science, 113, 4109-4120 (2000).
Special Function #1: Morphogenesis
Cell “coupled” through electrical signals – gap junctions.
1) Organize cells into functional domains.
* delimit populations of neuronal cells during spinal cord development.
1
2
J. Cell Science, 113, 4109-4120 (2000).
2) Healing in the epithelium.
* disruption of polarized layers = generation of guidance cues for cell migration.
* precursor cells migrate to site of injury, repair wound.
Special Function #2: Regeneration
Currents play a role in appendage regeneration:
* DC signal called “current of injury” is present in all animals, but is unique among
regenerating animals.
* peak voltage occurs at the time of maximum cell proliferation.
Inhibited
gap junctions
Adams D.S., Tissue
Engneering, 14, 1461–1468
(2008).
Special Function #2: Regeneration
Currents play a role in appendage regeneration:
* DC signal called “current of injury” is present in all animals, but is unique among
regenerating animals.
* peak voltage occurs at the time of maximum cell proliferation.
Inhibited
gap junctions
Adams D.S., Tissue
Engneering, 14, 1461–1468
(2008).
Sisken, B.F.,
Bioelectrochemistry and
Bioenergetics, 29, 121–126
(1992).
Region of positive voltage is larger than in non-regenerating animals (where current is
mostly slowly negative-going).
* current encircles the active end of stump, lasts for weeks and sufficient for inducing
regeneration.
Sites of Bioelectric-induced
Morphogenesis in Frog
Sites of regeneration due to
bioelectrical activity:
* misexpression of ion channel –
differences
in
developmental
morphogenesis (vs. control)
* modulated bioelectric cues =
changes in gene expression,
biochemistry during regenerative
morphogenesis
(morphogenetic
field).
Levin review, Figure 4.
Where does bioelectricity fit into
the analysis of physiological
systems?
Phase-space Approach
Levin review, Figure 5.
Phase-space: each component (measure) of the phenomenon (electrophysiology)
treated as an n-dimensional space.
Phase-space Approach
?
Levin review, Figure 5.
Furusawa and Kaneko, Biology Direct, 4: 17 (2009), Figure 1
Phase-space: each component (measure) of the phenomenon (electrophysiology)
treated as an n-dimensional space.
* phase space = all possible states a cell can take from one phenotype to another
(is it equivalent to a similar space created from genetic data?)
A “Curse of Orthogonality”?
Orthogonal: to be perpendicular, or at a
right angle (90̊) to:
* using one measurement type (mRNA),
cells appear to be different.
* using a seemingly parallel measure (Vmem),
results do not converge, but give another
answer.
Levin review, Figure 6.
A “Curse of Orthogonality”?
Orthogonal: to be perpendicular, or at a
right angle (90̊) to:
* using one measurement type (mRNA),
cells appear to be different.
* using a seemingly parallel measure (Vmem),
results do not converge, but give another
answer.
Less information overall than we would
expect:
* subadditive information with a linear
increase in variables.
Compare
to Bellman’s “curse of
dimensionality” (the more variables you
have, the harder problem becomes to solve).
Levin review, Figure 6.
What can translation tell us?
Fibroblast to excitable cell reprogramming
(+)
(+)
Stimulus
INSETS: IEEE Spectrum,
March 2011, 38-43
Transcriptionally
upregulated?
(+)
Presence of
mRNA
Production at
ribosome
Decay rate
(1/d)
Translationally
upregulated?