Freezing of Living Cells: Mathematical Models and Design of Optimal Cooling Protocols

AbstractActin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells.

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Mathematical models of radiation action on living cells: From the target theory to the modern approaches. A historical and critical review

Journal of Theoretical Biology ◽

10.1016/j.jtbi.2016.01.018 ◽

2016 ◽

Vol 394 ◽

pp. 93-101 ◽

Cited By ~ 32

Author(s):

Larry Bodgi ◽

Aurélien Canet ◽

Laurent Pujo-Menjouet ◽

Annick Lesne ◽

Jean-Marc Victor ◽

...

Keyword(s):

Mathematical Models ◽

Critical Review ◽

Living Cells ◽

Radiation Action ◽

Target Theory

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Mathematical model of tumour spheroid experiments with real-time cell cycle imaging

10.1101/2020.12.06.413856 ◽

2020 ◽

Author(s):

Wang Jin ◽

Loredana Spoerri ◽

Nikolas K. Haass ◽

Matthew J. Simpson

Keyword(s):

Mathematical Model ◽

Cell Cycle ◽

Mathematical Models ◽

Real Time ◽

Living Cells ◽

G1 Phase ◽

Tumour Spheroid ◽

Spheroid Growth ◽

M Phase ◽

The Mathematical Model

AbstractThree-dimensional (3D) in vitro tumour spheroid experiments are an important tool for studying cancer progression and potential drug therapies. Standard experiments involve growing and imaging spheroids to explore how different experimental conditions lead to different rates of spheroid growth. These kinds of experiments, however, do not reveal any information about the spatial distribution of the cell cycle within the expanding spheroid. Since 2008, a new experimental technology called fluorescent ubiquitination-based cell cycle indicator (FUCCI), has enabled real time in situ visualisation of the cell cycle progression. FUCCI labelling involves cells in G1 phase of the cell cycle fluorescing red, and cells in the S/G2/M phase of the cell cycle fluorescing green. Experimental observations of 3D tumour spheroids with FUCCI labelling reveal significant intratumoural structure, as the cell cycle status can vary with location. Although many mathematical models of tumour spheroid growth have been developed, none of the existing mathematical models are designed to interpret experimental observations with FUCCI labelling. In this work we extend the mathematical framework originally proposed by Ward and King (1997) to develop a new mathematical model of FUCCI-labelled tumour spheroid growth. The mathematical model treats the spheroid as being composed of three subpopulations: (i) living cells in G1 phase that fluoresce red; (ii) living cells in S/G2/M phase that fluoresce green; and, (iii) dead cells that do not fluoresce. We assume that the rates at which cells pass through different phases of the cell cycle, and the rate of cell death, depend upon the local oxygen concentration in the spheroid. Parameterising the new mathematical model using experimental measurements of cell cycle transition times, we show that the model can capture important experimental observations that cannot be addressed using previous mathematical models. Further, we show that the mathematical model can be used to quantitatively mimic the action of anti-mitotic drugs applied to the spheroid. All software required to solve the nonlinear moving boundary problem associated with the new mathematical model are available on GitHub.

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Fluorescence ratio imaging of dynamic intracellular ionic signals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102298 ◽

1988 ◽

Vol 46 ◽

pp. 42-43

Author(s):

R. Y. Tsien ◽

A. Minta ◽

M. Poenie ◽

J.P.Y. Kao ◽

A. Harootunian

Keyword(s):

Binding Sites ◽

Living Cells ◽

Molecular Engineering ◽

Ph Indicators ◽

Highly Sensitive ◽

Fluorescence Ratio Imaging ◽

Ratio Imaging ◽

Technical Advances ◽

Indicator Dyes ◽

Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102675 ◽

1988 ◽

Vol 46 ◽

pp. 118-119

Author(s):

K. Jacobson ◽

A. Ishihara ◽

B. Holifield ◽

F. Zhang

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Extended Period ◽

Dynamic Structure ◽

Living Cells ◽

Membrane Dynamics ◽

Molecular Cell Biology ◽

Image Averaging ◽

Single Living Cells ◽

Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122496 ◽

1992 ◽

Vol 50 (1) ◽

pp. 418-419

Author(s):

D. L. Taylor

Keyword(s):

Living Cells ◽

Cellular Level ◽

Molecular Techniques ◽

Cellular Functions ◽

Macromolecular Assemblies ◽

Cell Functions ◽

Molecular Events ◽

Yield Information ◽

Full Knowledge ◽

Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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4-dimensional, high-resolution imaging of living cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122484 ◽

1992 ◽

Vol 50 (1) ◽

pp. 416-417

Author(s):

Shinya Inoué

Keyword(s):

Light Microscope ◽

Living Cells ◽

Live Cells ◽

Video Tape ◽

Active Living ◽

High Resolution Imaging ◽

3 Dimensional ◽

Dynamic Architecture ◽

Resolution Imaging ◽

Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

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Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140889 ◽

1995 ◽

Vol 53 ◽

pp. 902-903

Author(s):

J. R. Kuhn ◽

M. Poenie

Keyword(s):

Mitotic Spindle ◽

Actin Filaments ◽

Cell Shape ◽

Dynamic Structure ◽

Living Cells ◽

Cytoskeletal Proteins ◽

Polarization Microscopy ◽

Video Enhancement ◽

Ultrastructural Studies ◽

Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

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Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123258 ◽

1992 ◽

Vol 50 (1) ◽

pp. 570-571

Author(s):

Robert Hard ◽

Gerald Rupp ◽

Matthew L. Withiam-Leitch ◽

Lisa Cardamone

Keyword(s):

Basal Body ◽

Untreated Control ◽

Beat Frequency ◽

Primary Cultures ◽

Living Cells ◽

Power Stroke ◽

Ultrastructural Changes ◽

Lung Cells ◽

Basal Bodies ◽

Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

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Microtubule Dynamics and Organelle Transport in Reticulomyxa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158510 ◽

1990 ◽

Vol 48 (3) ◽

pp. 194-195

Author(s):

Yih-Tai Chen ◽

Ursula Euteneuer ◽

Ken B. Johnson ◽

Michael P. Koonce ◽

Manfred Schliwa

Keyword(s):

Plasma Membrane ◽

Light Microscopy ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Microtubule Dynamics ◽

Model Systems ◽

Organelle Transport ◽

Biochemical Characteristics ◽

Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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