The identification of cells that form tubular structures in response to stress.

• Abstract
• Background
• Results
• Discussion
• Conclusion
• Methods
• References
• Table 1
• Figures

Thermodynamic and metabolic rates

Critics have postulated that straw cell structures are the artifacts from dead cells, the polymerization of membrane systems, or growth of protein crystals. Assuming that is the case, then the conversion from a spherical to straw cell is a simple physical deformation process. Therefore, the free energy needed to maintain spherical and tubular structures, built from the same amount of biological material, can be estimated. Following the Helfrich theory [16], the equilibrium shape of a thin membrane covering the vesicle or emulsion droplet is determined by the minimization of the Helmholtz free energy E_H (or, namely, the "shape energy") of the system

(1)

The first term in Eq. (1) is given by the pressure difference across the membrane Δp and the volume of the droplet V, while the second term is determined by the interfacial tension σ and surface area A. The integral term in Eq. (1) is often known as the free bending energy, where R₀ is the spontaneous radius of curvature, κ and is the mean and Gaussian curvature elastic constants, respectively. For a spherical structure, the two primary radii of curvature R₁ = R₂ = R, and the first two terms in Eq. (1) are cancelled, so we have

(2)

For a tubular structure with the inner radius a, outer radius b and length L, R₂ = 8, we have

(3)

From literature, the κ and of the lipid bilayers are in the range of (0.1–1.0) × 10^-19 J (Derek, 2006), as a result, Eq. (2) yields E_H~10^-18-10^-17 J per spherical cell. These values for carbohydrates, which are the major components of the straw cell wall, are not available. From the TEM measurements, the spherical cell has a typical radius R = 10 μm while the straw cell filaments, has a length of 3 cm, or 3.0 × 10⁴ μm, an inner radius a = 0.3 μm and an outer radius b = 0.5 μm (Figure 7). It is immediately noted that the exceptionally large aspect ratio (10⁴) of the straw cell causes the second term in Eq. (3) to dominate any other term in Eq. (2) and (3) containing κ and . In addition, the first term in Eq. (3) always contributes accumulatively to the shape energy. Considerable free energy, mainly in the form of the elastic energy, is needed to account for the remarkable spherical-to-tubular shape transformation, with the cell volume increased from 4.2 × 10³ to 2.6 × 10⁴ (μm³) and the cell surface area increased from 1.3 × 10³ to 1.8 × 10⁵ (μm²). We conclude that the tubular structure, as compared to the spherical cell, is heavily disfavored in free energy; which leads us to believe that the spontaneous polymerization of dead cell matrix to tubular structure is an unlikely event.

In reality, the straw cells cannot be simplified to a rod-shaped structure with an overall uniform composition. Not only do they have microscopic hair-like structures covering the surface (Fig.2I), but they also have differences in composition from the tubular center to the filamentous extensions (anisotropic and inhomogeneous) that makes a uniformity assumption untenable. During the spherical to tubular shape transformation, we observed changes in cell volume, surface area, and cell surface composition. The biological transformation we describe differs from a physical event where energy and material exchanges occur throughout the process.

During the reversion of a straw cell to a normal round cell, the tubular center (1 to 2 μm in diameter), houses organelles and grows by enlargement perpendicular to the elongated cell wall (Fig 3A) into a regular round shaped cell. Cellular metabolic rates are determined by the diffusion of ions and biomolecules [17,18] and are expected to be much slower in straw cells than spherical cells because of the narrow cylindrical geometry restricting the intracellular diffusion of solutes compared to a round shaped geometry [19]. The diffusion coefficient of K⁺ in the t-tubules of skeletal muscle fibers is anomalous and 27% less than its value in free solution [20]. We also observed a much slower growth rate in straw cells (approximately 10 to 15 days) during reversion to regular sized cells, while normal unstressed spherical cells take 2 days to duplicate. It is unclear how straw cells resume a normal round cellular morphology. It is also uncertain as to how ion channels operate during straw cell development.

Straw cell filaments have an outer diameter from 50 to 100 μm (Fig. 1C) and appear to have little cytoplasm (Fig 1D). This is in contrast to fungal apical growth where the elongation of hyphae is typically characterized with a much greater amount of cytoplasm and a distinct endoplasmic reticulum that consists of a network of tubules connected to the nuclear envelope [21,22]. Cell shape in bacteria is often defined by the distinct presence of peptidoglycan, a complex polymer built with glycan chains that are interconnected via peptide crossbridges [23]. Cells with a more complex shape, such as rod-shaped cells, exhibit an additional growth mode responsible for cell elongation [24]. It is unknown if straw cell assembly in regard to the cell wall is similar to bacteria, but glycoproteins and sulphated glucose polymers are primary constituents of the straw cell wall.

Indeed, straw cells can be independently observed in a 5 min experiment. This simply involves collecting any mammalian tissue fluid, placing 1 μL on a glass slide, and observing the droplet under a light microscope. As the droplet dries, the existence of straw cells and their connected filament networks are readily seen [see Additional file 1].

Proposed model of transformation

We propose that water activity sensors, present on the surface of mammalian cells, survey the hydration status of the environment and may be a type of membrane bound protein kinase(s). In plants, mitogen-activated protein (MAP) kinases [25-27] and phospohlipase D [28] have been upstream responders to the drought stress signaling pathway in Arabidopsis. A study of osmotic-stress-related proteins in rice has identified 12 specific proteins including kinases [29]. Water sensing may be a conserved mechanism throughout eukaryotes and mammalian cells may use similar MAP kinases for intracellular signaling. These sensors, when triggered, invoke a signaling cascade resulting in stress related transcriptional activity in the nucleus. As a result, we postulate that in mammalian cells, one of the potential protective responses involves a dramatic change in cell morphology resulting in the formation of straw cells.

Fragmentation of the CACO-2 nucleus (Figure 8, arrow), along with a synthesis of tubular wall was observed during the straw cell transformation process. The time-lapse images were obtained over the entire dehydration process using light microscopy and TEM. These images reveal the presence of polymers assembled along the nuclear membrane (arrows). These subunits are hypothesized to be the building material for the tubular cell wall, presumably, after they have cross-linked to each other.

In an attempt to identify the presence of the surface water activity sensors and subsequent proteins involved in the signal cascade, we used small molecules to inhibit straw cell transformation. Initial results indicated that this process could be manipulated and inhibited with naturally occurring compounds as well as synthetic compounds. For example, changes in membrane fluidity by salts and sugars (1%) disrupt the tubulFigure 8ar transformation; antibiotics (azithromysin) that inhibit protein synthesis interfered with the tubular transformation by producing filaments that were somewhat truncated.

Implication in human diseases

The large quantities of straw cells and their ubiquitous nature make them a potential unifying factor across a diversity of age related diseases. The conversion of cells into straw cells may be involved in the initial onset and progression of these diseases, the causes of which are still unknown. Moreover, straw cell formation may be a common physiological response in various types of human degenerative disease. Another area of human health where this phenomenon may play a role is tumor development and proliferation. The hydrophilic, mobile, detachment of straw cells may be a key component of metastasis. As such, hydrophilic straw cells from cancer cells may possibly move around in extracellular fluid and find new locations to grow.

There are many unanswered questions regarding the composition of these tubular cell structures found in a variety of mammalian tissues. For example, what suite of genes encodes the instructions governing the assembly of these tubular structures? What are the necessary molecules for making "unions" and "tees" in the filaments so as to form straw cell networks? Is straw cell transformation a conserved mechanism that mammalian cells use in response to the stress of dehydration and perhaps other stress inducing environmental factors? What is the physiological role of straw cell formation in vivo? How much dehydration occurs inside the body either locally or systemically?

Because the cellular transformation is reproducible in vitro in a 96-well plate, screening chemical libraries for inhibitory compounds to filamentous transformation is feasible and may result in the ability to control straw cell development in vivo. This may have implications in mediating degenerative diseases and tumor proliferation. For example, an In vitro assay using azithromysin at 10 ppm resulted in mammalian straw cells with both smaller straw cells and shortened filaments (Wu et al., unpublished data). One type of strategy could include selectively inhibiting protein kinases on the cell surface desensitizing the stress surveillance mechanism.