MORPHOLOGICAL AND FUNCTIONAL EXPRESSION INDUCED IN MAMMALIAN

CELLS BY GRAVITATIONAL AND MECHANICAL STRESS

Monica Monici

ASAcampus, ASA Research Division, Dept. of Clinical Physiopathology,

University of Florence

Physical Stresses and Cell Behaviour

Gravitational Stresses

Photomechanical Stress (Nd:YAG Laser)

Magnetic Fields

Mechanical forces are essential for tissue homeostasis. In particular, for the homeostasis of bone, cartilage and connective tissue.

Such tissues are not passively bearing the stress resulting from gravity, compression, and muscle-generated forces, but rather interplay with these dinamically. As a result, tissues modify their composition and mechanical properties

Gravitational/mechanical stress is the physical condition that exists within any cell as a result of deformation by external forces changing the pre-existing balance.

Chiquet et al.

Living cells are hard-wired by a continuous series of molecular struts (microtubules, cross-linked microfilament bundles), cables (contractile microfilaments), and ropes (intermediate filaments) in the form of a discrete CSK network.

by Ingber

Cytoskeleton organization

Cells use a particular form of architecture, known astensegrity, to organize and mechanically stabilize this CSK network.

Tensegrity structures (see geodesic architecture) havethe ability to carry a given load with a minimal amount of building materials.

Tensegrity structures stabilize themselves by balancing tension and compression forces.

by Ingber

CSK mechanically connects specific trans-membrane adhesion receptors on the cell surface to discrete contacts on the nucleus in the center of the cell.

by Ingber

Cells may sense mechanical stresses, including those due to gravity, through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the extracellular matrix and to other cells.

The cellular response to stress differs depending on the level of pre-stress (pre-existing tension) in the cytoskeleton.

by Ingber

IMMUNOFLUORESCENCE: Vimentin

Chondrocytes

Dendritic Cells

1xg 10xg

Tensegrity provides a way to focus mechanical energy on specificmolecular components and to integrate part and whole, therebytuning the entire cellular (and tissue) response.

Gravity acting on the whole organism is a major contributor to pre-stress within individual tissues.

When organisms are placed in microgravity, they experience an acute decrease in pre-stress on the macroscale which, due to the hierarchical organization of living tissues, should trickle down to produce corresponding changes in structure and mechanics at the cellular and molecular level.

by Ingber

A SUGGESTION:

To discard the concept of a single gravity-specific receptor molecule and to try of understanding gravity sensation in the context of the structural complexity of living cells and tissues. From this perspective, it is likely that in some cases the whole cell or even the whole tissue must be viewed as "the" gravity sensor.

BONE CELLS

Percentage of apoptotic cells

Comparison between controls at 1xg and samples exposed for 72 h to modelled hypogravity conditions

Cytofluorometric determination by AnnexinV assay

Apoptosis

0

20

40

60

80

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Gen

e ex

pres

sion

(a.u

.)

FAS FAS-L BAX

Control RPM

Gene expression screened in FLG 29.1 cells cultured for 72 h in a RPM. Comparison with control (1xg).

Osteoclastic precursors exposed to modelled weightlessness

Markers of osteoclasticdifferentiation

Protein (A) and gene (B) expressions screened in FLG 29.1 cells after 72 h of culturing in modelled hypogravity. Comparison with 1xg control

DENDRITIC CELLS

DENDRITIC CELLS AND THE CONTROL OF IMMUNITY

OpticalOptical MicroscopyMicroscopy (after 7 (after 7 daysdays))

ControlControl AstronautAstronaut (PM)(PM)

RTRT--PCRPCR

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60

80

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120

Ge

ne

exp

ress

ion

(a

.u.)

CD83 CD80 CD 86 CD40 NFkB

Astronaut DCs (PM) Control DCs

Expression of genes encoding for maturation markers

0.00.1

0.20.3

0.4

0.5

0.6

0.70.8

0.91.0

350 400 450 500 550 600 650 700 750

wavelength (nm)

inte

nsity

(a.u

.)

Control DCs

MultispectralMultispectral ImagingImagingAutofluorescenceAutofluorescence MicroscopyMicroscopy

AutofluorescenceAutofluorescence imaging imaging of immature of immature DCsDCs

AutofluorescenceAutofluorescenceMicrospectroscopyMicrospectroscopy

AutofluorescenceAutofluorescence spectrumspectrumof immature of immature DCsDCs

AutofluorescenceAutofluorescence microspectroscopymicrospectroscopy

0.00.10.20.30.40.50.60.70.80.91.0

350 400 450 500 550 600 650 700 750

wavelength (nm)

inte

nsity

(a.u

.)control DCs, day 6control DCs, day 7

AutofluorescenceAutofluorescence spectraspectra of of immature and mature control immature and mature control DCsDCs

AutofluorescenceAutofluorescence microspectroscopymicrospectroscopy

0.00.10.20.30.40.50.60.70.80.91.0

350 400 450 500 550 600 650 700 750

wavelength (nm)

inte

nsity

(a.u

.)Astronaut DCs (PM), day 6

Astronaut DCs (PM), day 7

Autofluorescence spectra of immature and mature astronaut DCs (PM)

CHONDROCYTES

IMMUNOFLUORESCENCE: Tubulin

Control 10xg Laser

IMMUNOFLUORESCENCE: Aggrecan

Control 10xg Laser

IMMUNOFLUORESCENCE: Collagen II

Control 10xg Laser

IMMUNOFLUORESCENCE: Sox 9

Control 10xg Laser

FIBROBLASTS

IMMUNOFLUORESCENCE: Cytoskeleton

Actin

Tubulin

Control Laser

IMMUNOFLUORESCENCE: Collagen I

Control Laser

IMMUNOFLUORESCENCE: Fibronectin

Control Laser Laser

FINAL REMARKS

Gravitational forces may be experienced by individual cells as aresult of stress-dependent changes that, in turn, alter extracellular matrix mechanics, cell shape, cytoskeletalorganization, or internal pre-stress in the cell-tissue matrix.

The body of the literature suggests that the effects of gravitational/mechanical stimuli is cell- and even function-specific.

Further research is needed in this field because the understanding of mechanotransduction at tissue and cell level can lead to:

• Efficient counteraction of the negative effects of microgravity by using mechanical stress

• Application of mechanical stress in modulating cell behaviourand tissue homeostasis: as an example, hypergravitational and photomechanical stress can increase the production of extracellular matrix molecules

Thanks to:

Marianne and Augusto Cogoli

Nicola Marziliano, Pietro Bernabei, Milena Paglierani

Roberto Vittori, Daniela Santucci

Lydia Bellik, Astrid Parenti, Fabrizio Ledda

Damiano Fortuna

Susanna Benvenuti, Mario Serio

Venere Basile, Giovanni Romano, Lucia Morbidelli,

Franco Fusi, Antonio Conti