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Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Apr 01, 2015

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Page 1: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

www.carleton.ca/~kbstorey

Page 2: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 3: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ADAPTATIONS TO COLDADAPTATIONS TO COLD

Below 0°C

Freeze Freeze AvoidanceAvoidance

Freeze Freeze ToleranceTolerance HibernationHibernation

Invertebrates

Some reptiles& amphibians

MigrationMigration

Mammals

Above 0°C

Others

Stay warmStay warm

Supercool

Page 4: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ADAPTATIONS TO COLDADAPTATIONS TO COLD

Below 0°C

Freeze Freeze AvoidanceAvoidance

Freeze Freeze ToleranceTolerance HibernationHibernation

Invertebrates

Some reptiles& amphibians

MigrationMigration

Mammals

Above 0°C

Others

Stay warmStay warm

Supercool

Page 5: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 6: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 7: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

MIGRATIONMIGRATION

Large mammals

Birds

Monarch Butterflies

Species able to book airline flights

Latitudinal & Altitudinal

Page 8: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Migrating Migrating DOWNDOWN

Page 9: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Fresh Water Fresh Water

Page 10: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Salt waterSalt water

Page 11: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ADAPTATIONS TO COLDADAPTATIONS TO COLD

Below 0°C

Freeze Freeze AvoidanceAvoidance

Freeze Freeze ToleranceTolerance HibernationHibernation

Invertebrates

Some reptiles& amphibians

MigrationMigration

Mammals

Above 0°C

Others

Stay warmStay warm

Supercool

Page 12: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Myotis lucifugus, little brown bat

Spermophilus tridecemlineatus,13-lined ground squirrel

Spermophilus richardsonii,Richardson’s ground squirrel

Page 13: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

• Seasonal phenomenon

• Pre-hibernation hyperphagia

• Gain up to 40% of body mass

• Need polyunsaturated fats

• Find hibernaculum: dark, near 0°C

Page 14: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

CELL PROCESSESCELL PROCESSES

• DNA/RNA synthesis• Protein synthesis • Fuel metabolism• Ion pumping • Work done

ATP turnover to <5% of normal

Page 15: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

METABOLIC RATE METABOLIC RATE DEPRESSION DEPRESSION

1. Slow Cell Processes

2. Use protein kinases (activate SAPKs)

3. Selective gene activation

Page 16: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

METABOLIC RATE METABOLIC RATE DEPRESSION DEPRESSION

• Protein Synthesis slows to 1% • Pumps & Channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated

• Gene ‘inactivation’

• Few Genes activated

Page 17: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

METABOLIC RATE METABOLIC RATE DEPRESSION DEPRESSION

• Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated

• Gene ‘inactivation’ (miRNA)

• Few Genes activated (1 % only)

Page 18: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

cDNA ARRAY SCREENINGcDNA ARRAY SCREENINGcontrol experimental

Page 19: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Diapause

Freezing

Anoxia

Hibernation

Page 20: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ADAPTATIONS TO COLDADAPTATIONS TO COLD

Below 0°C

Freeze Freeze AvoidanceAvoidance

Freeze Freeze ToleranceTolerance HibernationHibernation

Invertebrates

Some reptiles& amphibians

MigrationMigration

Mammals

Above 0°C

Others

Stay warmStay warm

Supercool

Page 21: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZE TOLERANT FREEZE TOLERANT ANIMALSANIMALS

• TERRESTRIAL INSECTS

• INTERTIDAL MOLLUSCS & BARNACLES

• AMPHIBIANS & REPTILES: - FROGS (6 species) - HATCHLING PAINTED TURTLES - GARTER SNAKES - LIZARDS (some)

Page 22: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 23: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Garter snake,Thamnophis

sirtalis

Page 24: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Painted turtle Painted turtle hatchlingshatchlings

Chrysemys pictaChrysemys pictamarginatamarginata

Page 25: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Box turtle, Box turtle, Terrapene carolinaTerrapene carolina

““OSCAR”OSCAR”

Page 26: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

GRAY TREE FROGHyla versicolor

Page 27: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

SPRING PEEPERPseudacris crucifer

CHORUS FROGPseudacris triseriata

Page 28: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

WOOD FROGRana sylvatica

Page 29: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

WOOD FROGRana sylvatica

Page 30: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

A WOOD FROG LIFE A WOOD FROG LIFE

SUMMERSUMMER - spent in the woods, eating & growing

AUTUMNAUTUMN - hide in insulated spots on forest floor

WINTERWINTER - freeze when hibernation site falls to about -2°C; survive frozen to -10°C

SPRINGSPRING - thaw & revive, move to woodland ponds

Mating & egg layingMating & egg laying - within 1 week in early spring

Eggs & tadpolesEggs & tadpoles - develop fast before temporary ponds dry out; metamorphosis in early summer

Page 31: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Frogs of various colours are numerous in those parts as far North as the latitude 61°….as the Winter approaches, they burrow under the moss, at a considerable distance from the water, where they remain in a frozen state till the Spring. I have frequently seen them dug up with the moss (when pitching tents in Winter) frozen as hard as ice; in which state the legs are as easily broken off as a pipe-stem, without giving the least sensation to the animal; but by wrapping them up in warm skins, and exposing them to a slow fire, they soon recover life…”. Samuel Hearne

A Journey from Prince of Wales’s fort in Hudson’s Bay to the Northern Ocean in the Years 1769-1772

Page 32: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

SURVIVING FREEZINGSURVIVING FREEZING

• Extracellular freezing only

• Up to 70% ofbody water frozen

• High ‘polyols’

• Acclimation required

• Glucose

• Glycerol

• Sorbitol

Page 33: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

WOOD FROG WOOD FROG CRYOPROTECTANTSCRYOPROTECTANTS

• Blood glucose rises from ~5 mM to 200-400 mM

• Glucose triggered by ice formation

• Made from liver glycogen (180 mg/g)

• Liver is ~12% of body mass

• Glucose distribution via Blood: Liver > Core organs > Periphery

Blood

Liver

Heart

Kidney

Muscle

Page 34: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

GLYCOGEN GLYCOGEN PHOSPHORYLASEPHOSPHORYLASE

Glycogen + Pi

kinase

Phos a Phos b

phosphatase

Glucose-1-P + glycogen (n-1) 0 2 5 30 60 2 3 1 2 3 1 3 1 3

4 min hours days min hours TIME OF FREEZING TIME OF THAW

LiverPhosphorylase a

Activity, U/g

Page 35: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

TO SURVIVE FREEZINGTO SURVIVE FREEZING• Alter metabolism to synthesize cryoprotectants (polyols, sugars)

• Defend against intracellular desiccation

• Suppress metabolic rate

ACCOMPLISHED BY:ACCOMPLISHED BY:

• Activate signaling enzymes in every cell

- ‘SAP’ kinases

- Role: reversible controls on cell processes

Up-regulate selected genesUp-regulate selected genes

Page 36: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Diapause

Freezing

Anoxia

Hibernation

Page 37: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZE INDUCED FREEZE INDUCED CHANGESCHANGES

• Gene ‘inactivation’• Protein Synthesis slows to 1% • Pumps & Channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated• Gene inactivation (miRNA)

• Few Genes activated

Page 38: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZE-INDUCED FREEZE-INDUCED GENES: WOOD FROGSGENES: WOOD FROGScDNA Library / Gene Chip • Only 1 % of genes “on” • The Unknowns: Fr10, Li16, FR47

Storey KB 2004. Strategies for exploration of freeze responsive gene

expression: advances in vertebrate freeze tolerance. Cryobiology 48, 134-145

Page 39: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

THE UKNOWNS :THE UKNOWNS :Li16, FR10, FR47Li16, FR10, FR47

• Novel gene sequences discovered by cDNA library screening

• Genes moved to other cell types

• Genomic sequences now known

• On-Off Regulation: Protein Kinases

• Proteins are biomanufactured in our lab

• Non freeze tolerant cells can be transformed

Page 40: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FUNCTION OF THE FUNCTION OF THE UNKNOWN PROTEINSUNKNOWN PROTEINS

• Express genes in cells in culture - Li16, FR10 - insect or mammal cells

• Expression of Li16 & FR10 protects cultured cells from freezing damage

• Li16 is intracellular

• FR10 is exported

• Both bind to membranes

Li16 FR10 NT none

% C

ell s

urv

ival

0

20

40

60

80

100

Survival of -6oC freezingin the presence of

overexpressed proteins

Page 41: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Unique Animal Stress ModelUnique Animal Stress Model

Vertebrate whole-body freeze tolerance

Tissue cryopreservation

Tolerance of extreme ischemia and hyperglycemia

Page 42: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

CRYOPRESERVED CRYOPRESERVED TISSUESTISSUES

• SPERM• EMBRYOS• SKIN• CORNEA• VEINS• BLOOD CELLS• HEART VALVES

• TEETH, BONE• BONE MARROW• PANCREATIC TISSUE• THYROID TISSUE• PARATHYROID TISSUE• FETAL TISSUES (some)• **RAT LIVER**

Page 43: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ORGANS FOR TRANSPLANTORGANS FOR TRANSPLANT1. Scientific Solutions1. Scientific Solutions

A. IMMEDIATE:A. IMMEDIATE: extend the viability of removed organs by hours/daysB. FUTURE: B. FUTURE: - freeze organs to create organ banks - stem cell research - grow new organsC. FAR FUTURE:C. FAR FUTURE: - cloning of tissues (one cell --> organ) - artificial tissues (from non-cell sources)D. XENOTRANSPLANTSD. XENOTRANSPLANTS - Dangers and risks?E. Clone “NEAR-HUMANS” for parts:E. Clone “NEAR-HUMANS” for parts: - Society plus science (+/- embryos) - Have your own clone, just in case? - The rights of a clone?

Page 44: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

ORGANS FOR TRANSPLANTORGANS FOR TRANSPLANT

2. Society Solutions: Dollars, Science, Morals2. Society Solutions: Dollars, Science, Morals

A. SELL ORGANS:A. SELL ORGANS: $$ from rich to poor people Organs from poor to rich people Morally correct? How to regulate?

B. Get organs by “PRESUMED CONSENT”B. Get organs by “PRESUMED CONSENT” - Will doctors revive or harvest? - How dead do you have to be? - Religious / spiritual implications

Tens of thousands wait for a few organs - who decides? - should you be able to pay for an organ?

Page 45: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

THE FUTURE ??THE FUTURE ??

Page 46: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 47: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 48: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.
Page 49: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZING HUMANSFREEZING HUMANSDOES IT WORK ?

A. Liquid Nitrogen Storage ( -196°C)

- frogs only to -20°C (cell destruction)

- fragility/crush (neurons)

B. Frozen Liquid Expands !

C. Bits and Bobs ……….

D. Time to Preservation (oxygen lack, neurons)

E. You’ve paid UPFRONT for “forever” !

F. Legal implications (thawed by your kids)

Page 50: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZING HUMANSFREEZING HUMANSIs it correct to freeze humans and then

bring them back in the future for “eternal life”?

A. Who would be chosen for this (costly) procedure?

B. How would we pay for re-animation and re-integration into society? - for 20 subjects - for 2000 subjects - for 2 billion subjects

C. Spiritual / Religious implications

D. Legal implications

Page 51: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

www.carleton.ca/~kbstorey

Dr. Ken StoreyInstitute of BiochemistryCarleton UniversityOttawa, Canada

Page 52: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

Estivation

Diapause

Freezing

Anoxia

Hibernation

Page 53: Www.carleton.ca/~kbstorey. ADAPTATIONS TO COLD Below 0°C Freeze FreezeAvoidanceFreezeTolerance Hibernation Invertebrates Some reptiles & amphibians.

FREEZE TOLERANCEFREEZE TOLERANCE• J. STOREY• D. McNALLY• J. MacDONALD• T. CHURCHILL• S. GREENWAY• C. HOLDEN• S. WU

• A. DeCROOS• L. ZHENHONG• J. DU• Q. CAI• F. SCHUELER• S. BROOKS• B. RUBINSKY• R. BROOKS

www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey