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

Electrical Field (dipole with no

intermediate barrier).

Membrane potential (dipole with

selective permeability through a

barrier).

Cellular bioelectric potential expressed in terms of

dipoles:

Membrane Potential vs. Electrical Fields

Electrical Field (dipole with no

intermediate barrier).

Membrane potential (dipole with

selective permeability through a

barrier).

Cellular bioelectric potential expressed in terms of

dipoles:

Ion concentrations form

gradients between inside

and outside of membrane

via ion channels.

Vmem

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

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.

Fluxes: changes in the flow of ions over time.

* channels open and close – introduces

selective permeability.

* “bursty” response functions (e.g. action

potentials).

Transport of Electrical Signals

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.

Sundelacruz, Levin, and Kaplan paper, Table 1

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:

Hyperpolarization:

Negative-going or negative

membrane potential.

* inhibits rise of action

potential.

Depolarization:

Positive-going or positive

membrane potential.

COURTESY: http://bioserv.fiu.edu/~walterm/GenBio2004/

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.

* further hyperpolarization drives G1/S transition.

2) Glioma model: inwardly-rectifying Kir4.1 channel

during proliferation:

* expressed specifically in glial-differentiated

astrocytes.

COURTESY:

http://wwwsciencephoto.com

Codes for

protein Kv11.1:

cardiac rhythm

and cancer

establisher.

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 Imaging (e.g. FRET) Electrophysiology Dye Imaging (e.g. FRET) COURTESY: Pacific Northwest National Labs

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).

COURTESY: Nature Reviews Molecular

Cell Biology 5, 614-625 (2004)

* K+ channel subunits = 4

TM, 2 P domains.

* TASK 2p phenotype leaky

when closed (mutant).

* expression = death in

proper conditions (e.g. pH).

* important in 1:1 granular-

Purkinje 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”.

* aggregate cellular behavior gives

us the type and number of each cell

type. Sundelacruz, Levin, and Kaplan review, Figure 1

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 matrix (ECM) dynamics.

* chemical effects (microenvironment).

Signaling has a multiplicative effect (combinatorial).

Michael Levin’s “formula” for development and

regeneration (Levin review, Figure 1)

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.

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.

1 2

J. Cell Science, 113, 4109-4120 (2000).

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.

Adams D.S., Tissue

Engneering, 14, 1461–1468

(2008). Inhibited

gap junctions

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.

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.

Adams D.S., Tissue

Engneering, 14, 1461–1468

(2008).

Sisken, B.F.,

Bioelectrochemistry and

Bioenergetics, 29, 121–126

(1992).

Inhibited

gap junctions

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

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?)

Furusawa and Kaneko, Biology Direct, 4: 17 (2009), Figure 1 Levin review, Figure 5.

?

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?

Transcriptionally

upregulated?

Translationally

upregulated?

Fibroblast to excitable cell reprogramming

Stimulus Production at

ribosome

Presence of

mRNA

Decay rate

(1/d)

(+) (+) (+)

INSETS: IEEE Spectrum,

March 2011, 38-43