Separation of preterm infection model from normal pregnancy in mice using texture analysis of second harmonic generation images.

This paper presents an image processing system to distinguish a lipopolysaccharide (LPS) infection model of preterm labor from normal mouse pregnancy using Second Harmonic Generation (SHG) images of mouse cervix. Two classes of SHG images are considered: images from mice in which premature birth was caused by intrauterine LPS administration and images from normal pregnant mice. A wide collection of image texture features consisting of co-occurrence matrix-based, granulometry-based and wavelet-based are examined. The results obtained indicate that the combination of co-occurrence-based and granulometry-based textures features provides the most effective texture set for separating these two classes of images.

A statistical approach for intensity loss compensation of confocal microscopy images.

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area.

Classical biochemistry is founded on several assumptions valid in dilute aqueous solutions that are often extended without question to the interior milieu of intact cells. In the first section of this chapter, we present these assumptions and briefly examine the ways in which the cell interior may depart from the conditions of an ideal solution. In the second section, we summarize experimental evidence regarding the physical properties of the cell cytoplasm and their effect on the diffusion and binding of macromolecules and vesicles. While many details remain to be worked out, it is clear that the aqueous phase of the cytoplasm is crowded rather than dilute, and that the diffusion and partitioning of macromolecules and vesicles in cytoplasm is highly restricted by steric hindrance as well as by unexpected binding interactions. Furthermore, the enzymes of several metabolic pathways are now known to be organized into structural and functional units with specific localizations in the solid phase, and as much as half the cellular protein content may also be in the solid phase.

Mechanism of Ca2+-dependent nuclear accumulation of calmodulin.

The intracellular Ca2+ receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca2+-dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca2+-dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca2+-dependence and affinity. At high [Ca2+], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.

Properties of filament-bound myosin light chain kinase.

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.

Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav.

Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

Viscoelastic response of fibroblasts to tension transmitted through adherens junctions.

Cytoplasmic deformation was monitored by observing the displacements of 200-nm green fluorescent beads microinjected into the cytoplasm of Swiss 3T3 fibroblasts. We noted a novel protrusion of nonruffling cell margins that was accompanied by axial flow of beads and cytoplasmic vesicles as far as 50 microm behind the protruding plasma membrane. Fluorescent analog cytochemistry and immunofluorescence localization of F-actin, alpha-actinin, N-cadherin, and beta-catenin showed that the protruding margins of deforming cells were mechanically coupled to neighboring cells by adherens junctions. Observations suggested that protrusion resulted from passive linear deformation in response to tensile stress exerted by centripetal contraction of the neighboring cell. The time dependence of cytoplasmic strain calculated from the displacements of beads and vesicles was fit quantitatively by a Kelvin-Voight model for a viscoelastic solid with a mean limiting strain of 0.58 and a mean strain rate of 4.3 x 10(-3) s(-1). In rare instances, the deforming cell and its neighbor spontaneously became uncoupled, and recoil of the protruding margin was observed. The time dependence of strain during recoil also fit a Kelvin-Voight model with similar parameters, suggesting that the kinetics of deformation primarily reflect the mechanical properties of the deformed cell rather than the contractile properties of its neighbor. The existence of mechanical coupling between adjacent fibroblasts through adherens junctions and the viscoelastic responses of cells to tension transmitted directly from cell to cell are factors that must be taken into account to fully understand the role of fibroblasts in such biological processes as wound closure and extracellular matrix remodeling during tissue development.

Binding of myosin light chain kinase to cellular actin-myosin filaments.

Myosin light chain kinase binds to the actomyosin-containing filaments in smooth and nonmuscle cells. However, the region of the kinase necessary for this high affinity binding in vivo is not known, although it has been proposed that the N and C termini bind to actin and myosin in vitro, respectively. Truncated myosin light chain kinases containing the catalytic core and calmodulin-binding domain but lacking N (amino acids 1-655) and/or C (amino acids 1004-1147) termini were expressed in the baculovirus system and purified. All enzymes were catalytically active and Ca2+/calmodulin-dependent. The C-terminal truncated myosin light chain kinase bound to detergent-washed smooth muscle contractile proteins similar to recombinant full-length myosin light chain kinase or enzyme purified from smooth muscle. The apparent affinity of the full-length kinase was greater for the actomyosin-containing filaments with associated proteins than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. In contrast, truncations at the N terminus alone or at both N and C termini resulted in no significant binding. Similar effects were observed by two other assays: binding of fluorescently labeled myosin light chain kinases to actin-containing stress fibers in detergent-treated fibroblasts and localization of fluorescently labeled kinases after microinjection into primary smooth muscle cells in culture. The full-length and the C-terminal truncated myosin light chain kinases, but not myosin light chain kinases truncated at the N terminus or both N and C termini, associated with filaments in cells. Thus, the N terminus and not the C terminus of myosin light chain kinase is necessary for high affinity binding to actomyosin-containing filaments in smooth and nonmuscle cells.

Tracer diffusion through F-actin: effect of filament length and cross-linking.

We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity.

Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains.

Subdomains of the cytoplasmic volume in tissue culture cells exclude large tracer particles relative to small. Evidence suggests that exclusion of the large particles is due to molecular sieving by the dense meshwork of microfilaments found in these compartments, but exclusion as a result of the close apposition of the dorsal and ventral plasma membrane of the cell in these regions has not been ruled out conclusively. In principle, these two mechanisms can be distinguished by the dependence of exclusion on tracer particle size. By fluorescence ratio imaging we have measured the partition coefficient (P/PO) into excluding compartments for tracer particles ranging in radius from 1 to 41 nm. The decay of P/PO as a function of particle radius is better fitted by three molecular sieving models than by a slit pore model. The sieving models predict a percolation cutoff radius of the order of 50 nm for partitioning into excluding compartments.

Ca(2+)-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells.

A key assumption of most models for calmodulin regulation of smooth and non-muscle contractility is that calmodulin is freely diffusible at resting intracellular concentrations of free Ca2+. However, fluorescence recovery after photobleaching (FRAP) measurements of three different fluorescent analogs of calmodulin in cultured bovine tracheal smooth muscle cells suggest that free calmodulin may be limiting in unstimulated cells. Thirty-seven % of microinjected calmodulin is immobile by FRAP and the fastest recovering component has an effective diffusion coefficient 7-fold slower than a dextran of equivalent size. Combining the FRAP data with extraction data reported in a previous paper (Tansey, M., Luby-Phelps, K., Kamm, K.E., and Stull, J.T. (1994) J. Biol. Chem. 269, 9912-9920), we estimate that at most 5% of total endogenous calmodulin in resting smooth muscle cells is unbound (freely diffusible). Examination of the Ca2+ dependence of calmodulin mobility in permeabilized cells reveals that binding persists even at intracellular Ca2+ concentrations as low as 17 nM. When Ca2+ is elevated to between 450 nM and 3 microM, some of the bound calmodulin is released, as indicated by an increase in the effective diffusion coefficient and the percent mobile fraction. At higher Ca2+, calmodulin becomes increasingly immobilized. In about 50% of the cell population, clamping Ca2+ at micromolar levels results in translocation of cytoplasmic calmodulin to the nucleus. The compartmentalization and complex dynamics of calmodulin in living smooth muscle cells have profound implications for understanding how calmodulin regulates contractility in response to extracellular signals.

Ca(2+)-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells.

Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain. Addition of KN-62 or a synthetic peptide CK II, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase II activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodamine-calmodulin in intact tracheal smooth muscle cells.

Physical properties of cytoplasm.

The physical properties of cytoplasm differ considerably from dilute aqueous solutions. Recent research has improved our understanding of the properties of the fluid phase and provided a more detailed picture of cytoarchitecture and its relation to cytomechanics. Several recent holistic models indicate novel directions for future research.

Cytoarchitecture of size-excluding compartments in living cells.

By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from otherwise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent non-excluding domains. The height of the distal excluding compartments was generally < or = 50% of the height in the adjacent non-excluding compartment. An electron-dense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin nonmuscle filamin (ABP280) and alpha-actinin. F-actin and ABP280, but not alpha-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the trans-Golgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.

Gap junction communication modulates [Ca2+]i oscillations and enzyme secretion in pancreatic acini.

Global (all cells in an acinus) and focal (1-2 out of 10-15 cells) stimulation of pancreatic acini with bombesin or t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Asp-2-phenylethyl ester (CCKJ) together with modulation of gap junction (GJ) permeability by octanol and NO2- was used to study the role of GJ permeability in controlling [Ca2+]i oscillations and enzyme secretion. GJ permeability was quantitated by measuring fluorescence recovery after photobleaching. Octanol at 0.5 mM markedly reduced, whereas 15 mM NO2- increased GJ permeability. Focal application of bombesin caused synchronized oscillations in the entire acinus, whereas global stimulation resulted in asynchronous oscillations. Increasing GJ permeability with NO2- had no effect on bombesin-evoked [Ca2+]i oscillations. Octanol inhibited ongoing oscillations evoked by focal or global bombesin stimulation. However, when GJ were blocked prior to stimulation, subsequent global stimulation with bombesin induced long-lasting oscillations in all cells. Re-establishing GJ communication for as little as 37.5 s conferred GJ dependence on the order and time of [Ca2+]i spiking evoked by global bombesin stimulation. Focal and global stimulation with CCKJ gave different patterns of [Ca2+]i oscillations. However, in contrast to bombesin, inhibition of GJ with octanol had no effect on oscillations induced by global CCKJ stimulation. Increasing GJ permeability with NO2- synchronized CCKJ-stimulated oscillations by equalizing the amplitude and increasing the frequency in all cells within an acinus. These observations suggest that amplitude and frequency of [Ca2+]i oscillations can be regulated independently of each other, and that GJ permeable molecules modulate the frequency of [Ca2]i oscillation in an agonist-specific manner. Regardless of the agonist, increasing the frequency of oscillations by modulation of GJ permeability correlated with an increased enzyme secretion.

A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.

Effect of cytoarchitecture on the transport and localization of protein synthetic machinery.

The emerging picture of cytoarchitecture imposes constraints on the transport and localization of several components of the protein synthetic machinery. The range in which "free" polysomes can diffuse through the cytoplasm may be restricted to about 50 nm due to obstruction by cytoskeletal barriers. Individual ribosomes and large transcripts will diffuse at least 4-10 times slower in cytoplasm than in dilute aqueous solution and may be sterically excluded from some cytoplasmic domains. The transport of these components from the nucleus to the cell periphery may be restricted to microtubule-containing channels that traverse the excluding domains. In the peripheral cytoplasm, mitochondria, endoplasmic reticulum, and other membrane-bound organelles are found only in nonexcluding channels, while actin, nonmuscle filamin (ABP280), and fodrin are concentrated in excluding domains. This suggests that the cytoplasmic volume may be functionally compartmentalized by local differentiations of cytoarchitecture. Excluding compartments may play a structural role, while nonexcluding compartments are the site of vesicle traffic and protein synthesis.

Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm.

We have previously reported that self-diffusion of inert tracer particles in the cytoplasm of living Swiss 3T3 cells is hindered in a size-dependent manner (Luby-Phelps, K., D.L. Taylor, and F. Lanni. 1986. J. Cell Biol. 102:2015-2022; Luby-Phelps, K., P.E. Castle, D.L. Taylor, and F. Lanni. 1987. Proc Natl. Acad. Sci. USA. 84:4910-4913). Lacking a theory that completely explains our data, we are attempting to understand the molecular architecture responsible for this phenomenon by studying tracer diffusion in simple, reconstituted model systems. This report contains our findings on tracer diffusion in concentrated solutions of Ficoll 70 or Ficoll 400, in solutions of entangled F-actin filaments, and in solutions of entangled F-actin containing a background of concentrated Ficoll particles or concentrated bovine serum albumin (BSA). A series of size-fractionated fluorescein-Ficolls were used as tracer particles. By fluorescence recovery after photobleaching (FRAP), we obtained the mean diffusion coefficients in a dilute, aqueous reference phase (Do), the mean diffusion coefficients in the model matrices (D), and the mean hydrodynamic radii (RH) for selected tracer fractions. For each model matrix, the results were compared with similar data obtained from living cells. As in concentrated solutions of globular proteins (Luby-Phelps et al., 1987), D/Do was not significantly size-dependent in concentrated solutions of Ficoll 400 or Ficoll 70. In contrast, D/Do decreased monotonically with increasing RH in solutions of F-actin ranging in concentration from 1 to 12 mg/ml. This size dependence was most pronounced at higher F-actin concentrations. However, the shape of the curve and the extrapolated value of D/Do in the limit, RH----O did not closely resemble the cellular data for tracers in the same size range (3 less than RH less than 30 nm). In mixtures of F-actin and Ficoll or F-actin and BSA, D/Do was well approximated by D/Do for the same concentration of F-actin alone multiplied by D/Do for the same concentrations of Ficoll or BSA alone. Based on these results, it is possible to model the submicroscopic architecture of cytoplasm in living cells as a densely entangled filament network (perhaps made up of F-actin and other filamentous structures) interpenetrated by a fluid phase crowded with globular macromolecules, which in cytoplasm would be primarily proteins.