Microfabricated post-array-detectors (mPADs): an approach to isolate mechanical forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 microm in diameter, 10 microm tall, and are configured in a regular array with 9 microm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

An array of microfabricated pillars to study cell migration

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.     

A novel patterned magnetic micropillar array substrate for analysis of cellular mechanical responses

Traction forces generated at cellular focal adhesions (FAs) play an essential role in regulating various cellular functions. These forces (1–100 nN) can be measured by observing the local displacement of a flexible substrate upon which cells have been plated. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces. A tool capable of applying a force to FAs independently, by actively moving the micropillars, should become a powerful tool to delineate the cellular mechanotransduction mechanisms. Here, we developed a patterned magnetic micropillar array PDMS substrate that can be used for the mechanical stimulation of cellular FAs and the measurement of associated traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Iron particles were embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field, which also controlled their location on the substrate. Applying a magnetic field of 0.3 T bent the pillars by ∼4 µm and allowed transfer of external forces to the actin cytoskeleton through FAs formed on the pillar top. Using this approach, we investigated the traction force changes in cultured aortic smooth muscle cells (SMCs) after local compressive stimuli to release cell pretension. The mechanical responses of SMCs were roughly classified into two types: almost a half of the cells showed a little decrease of traction force at each pillar following compressive stimulation, although cell area increased significantly; and the rest showed the opposite, with increased forces and a simultaneous decrease in area. The traction forces of SMCs fluctuated markedly during the local compression. The root mean square of traction forces significantly increased during the compression, and returned to the baseline level after its release. These results suggest that the fluctuation of forces may be caused by active reorganization of the actin cytoskeleton and/or its dynamic interaction with myosin molecules. Thus, our magnetic micropillar substrate would be useful in investigating the mechanotransduction mechanisms of cells.     

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness,Nuclear Volume,Contractility, and Cell Stiffness

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.     

Substrate Deformation Predicts Neuronal Growth Cone Advance

Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 10⁰–10² nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 μm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 μm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.     

Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.     

Traction Forces of Endothelial Cells under Slow Shear Flow

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.     

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.     

Factors Influencing the Ability of Listeria monocytogenes To Pass through a Membrane Filter by Active Infiltration

Listeria monocytogenes infiltrated the reticulate structure of a membrane filter and passed through a filter with pore sizes of 0.45 μm and 0.2 μm in 6 to 24 h and 5 to 6 days, respectively. Flagellar motility and expansive pressure generated by the growing bacterial population were indicated as the driving forces of infiltration.     

Nonlinear Strain Stiffening Is Not Sufficient to Explain How Far Cells Can?Feel on Fibrous Protein Gels

Recent observations suggest that cells on fibrous extracellular matrix materials sense mechanical signals over much larger distances than they do on linearly elastic synthetic materials. In this work, we systematically investigate the distance fibroblasts can sense a rigid boundary through fibrous gels by quantifying the spread areas of human lung fibroblasts and 3T3 fibroblasts cultured on sloped collagen and fibrin gels. The cell areas gradually decrease as gel thickness increases from 0 to 150 μm, with characteristic sensing distances of >65 μm below fibrin and collagen gels, and spreading affected on gels as thick as 150 μm. These results demonstrate that fibroblasts sense deeper into collagen and fibrin gels than they do into polyacrylamide gels, with the latter exhibiting characteristic sensing distances of <5 μm. We apply finite-element analysis to explore the role of strain stiffening, a characteristic mechanical property of collagen and fibrin that is not observed in polyacrylamide, in facilitating mechanosensing over long distances. Our analysis shows that the effective stiffness of both linear and nonlinear materials sharply increases once the thickness is reduced below 5 μm, with only a slight enhancement in sensitivity to depth for the nonlinear material at very low thickness and high applied traction. Multiscale simulations with a simplified geometry predict changes in fiber alignment deep into the gel and a large increase in effective stiffness with a decrease in substrate thickness that is not predicted by nonlinear elasticity. These results suggest that the observed cell-spreading response to gel thickness is not explained by the nonlinear strain-stiffening behavior of the material alone and is likely due to the fibrous nature of the proteins.     

E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior.     

Microstamped Petri Dishes for Scanning Electrochemical Microscopy Analysis of Arrays of Microtissues

While scanning electrochemical microscopy (SECM) is a powerful technique for non-invasive analysis of cells, SECM-based assays remain scarce and have been mainly limited so far to single cells, which is mostly due to the absence of suitable platform for experimentation on 3D cellular aggregates or microtissues. Here, we report stamping of a Petri dish with a microwell array for large-scale production of microtissues followed by their in situ analysis using SECM. The platform is realized by hot embossing arrays of microwells (200 μm depth; 400 μm diameter) in commercially available Petri dishes, using a PDMS stamp. Microtissues form spontaneously in the microwells, which is demonstrated here using various cell lines (e.g., HeLa, C2C12, HepG2 and MCF-7). Next, the respiratory activity of live HeLa microtissues is assessed by monitoring the oxygen reduction current in constant height mode and at various distances above the platform surface. Typically, at a 40 μm distance from the microtissue, a 30% decrease in the oxygen reduction current is measured, while above 250 μm, no influence of the presence of the microtissues is detected. After exposure to a model drug (50% ethanol), no such changes in oxygen concentration are found at any height in solution, which reflects that microtissues are not viable anymore. This is furthermore confirmed using conventional live/dead fluorescent stains. This live/dead assay demonstrates the capability of the proposed approach combining SECM and microtissue arrays formed in a stamped Petri dish for conducting cellular assays in a non-invasive way on 3D cellular models.     

Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds

We determined whether the molecular structures through which force is applied to receptor–ligand pairs are tuned to optimize cell adhesion under flow. The adhesive tethers of our model system, Escherichia coli, are type I fimbriae, which are anchored to the outer membrane of most E. coli strains. They consist of a fimbrial rod (0.3–1.5 μm in length) built from a helically coiled structural subunit, FimA, and an adhesive subunit, FimH, incorporated at the fimbrial tip. Previously reported data suggest that FimH binds to mannosylated ligands on the surfaces of host cells via catch bonds that are enhanced by the shear-originated tensile force. To understand whether the mechanical properties of the fimbrial rod regulate the stability of the FimH–mannose bond, we pulled the fimbriae via a mannosylated tip of an atomic force microscope. Individual fimbriae rapidly elongate for up to 10 μm at forces above 60 pN and rapidly contract again at forces below 25 pN. At intermediate forces, fimbriae change length more slowly, and discrete 5.0 ± 0.3–nm changes in length can be observed, consistent with uncoiling and coiling of the helical quaternary structure of one FimA subunit at a time. The force range at which fimbriae are relatively stable in length is the same as the optimal force range at which FimH–mannose bonds are longest lived. Higher or lower forces, which cause shorter bond lifetimes, cause rapid length changes in the fimbria that help maintain force at the optimal range for sustaining the FimH–mannose interaction. The modulation of force and the rate at which it is transmitted from the bacterial cell to the adhesive catch bond present a novel physiological role for the fimbrial rod in bacterial host cell adhesion. This suggests that the mechanical properties of the fimbrial shaft have codeveloped to optimize the stability of the terminal adhesive under flow.     

Traction forces generated by locomoting keratocytes

Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be approximately 2 x 10(-8) N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell- substratum contact.     

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida,Antarctica

The anoxic and freezing brine that permeates Lake Vida''s perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.     

Using Femtosecond Laser to Create Customized Corneal Flaps for Patients with Low and Moderate Refractive Error Differing in Corneal Thickness

This study is designed to evaluate the visual outcomes, accuracy, and predictability of corneal flaps with different thicknesses created by 60-kHz femtosecond laser according to different corneal thicknesses in the patients with low and moderate refractive error. A total of 182 eyes were divided according to the central corneal thickness (470μm–499 μm in Group A, 500μm–549 μm in Group B, and 550μm–599 μm in Group C) and underwent femtosecond laser-assisted LASIK for a target corneal flap thickness (100 μm for Group A, 110 μm for Group B, and 120 μm for Group C). Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and refractive status were examined. The flap thickness of each eye was measured by anterior segment optical coherence tomography (AS-OCT) on 30 points at 1-month follow-up to assess the accuracy and predictability. Postoperatively, at least 75% of eyes had a UDVA of 20/16 or better, less than 2% of eyes lost one line, over 30% of eyes gained one or more lines in CDVA, at least 95% of eyes had astigmatism of less than 0.25 D, all eyes achieved a correction within ±1.00 D from the target spherical equivalent refraction. The visual and refractive outcomes did not differ significantly in all groups (P >0.05). The mean flap thickness was 100.36± 4.32 μm (range: 95–113 μm) in Group A, 111.64 ± 3.62 μm (range: 108–125 μm) in Group B, and 122.32 ± 2.88 μm (range: 112–128 μm) in Group C. The difference at each measured point among the three groups was significant (P < 0.05). The accuracy and predictability were satisfactory in all three groups. In conclusion, this customized treatment yielded satisfactory clinical outcomes with accurate and predictable flap thickness for patients with low and moderate refractive error.     

Activity Measurements of Planktonic Microbial and Microfouling Communities in a Eutrophic Estuary

[³H]thymidine incorporation, the rate of reduction of iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA were adapted to measure the activity of attached and unattached microbial assemblages of Bayboro Harbor, Fla. Activity measurements by [³H]thymidine incorporation were made of cells attached to polystyrene culture dishes, in unfiltered water samples, and in the <1-μm-filtered fraction. In most cases, the activity of attached cells was greater than that of unattached cells either in unfiltered water samples or in the <1-μm fraction. The calculated thymidine incorporation rates for cells in the >1-μm fraction were higher than those for cells either in unfiltered water or in the <1-μm-filtered fraction. By the rate of reduction of INT to INT formazan normalized to DNA and by ATP-to-DNA ratios, attached cells were also more active than cells in unfiltered water samples. These results indicate that the microenvironment afforded by attachment is a more beneficial habitat for microbial growth. Reasons for greater activity by natural populations of attached bacteria are discussed.     

High resolution traction force microscopy based on experimental and computational advances

Cell adhesion and migration crucially depend on the transmission of actomyosin-generated forces through sites of focal adhesion to the extracellular matrix. Here we report experimental and computational advances in improving the resolution and reliability of traction force microscopy. First, we introduce the use of two differently colored nanobeads as fiducial markers in polyacrylamide gels and explain how the displacement field can be computationally extracted from the fluorescence data. Second, we present different improvements regarding standard methods for force reconstruction from the displacement field, which are the boundary element method, Fourier-transform traction cytometry, and traction reconstruction with point forces. Using extensive data simulation, we show that the spatial resolution of the boundary element method can be improved considerably by splitting the elastic field into near, intermediate, and far field. Fourier-transform traction cytometry requires considerably less computer time, but can achieve a comparable resolution only when combined with Wiener filtering or appropriate regularization schemes. Both methods tend to underestimate forces, especially at small adhesion sites. Traction reconstruction with point forces does not suffer from this limitation, but is only applicable with stationary and well-developed adhesion sites. Third, we combine these advances and for the first time reconstruct fibroblast traction with a spatial resolution of ∼1 μm.     

Margollus bokanicus n. sp. from Iran,the Fourth Species of a Rare Nematode Genus (Dorylaimida,Tylencholaimellidae)

Margollus bokanicus n. sp., collected from natural habitats in Khorasaneh district, Bokan, West Azarbaijan province, Iran, is described. Morphological and morphometric data are provided as well as drawings and light microscopy illustrations. The new species is characterized by a medium size body length (0.60 to 0.73 mm), labial and postlabial sclerotizations, lip region 7-μm wide, offset by constriction and long neck (167 to 207 μm), long pharyngeal basal bulb (27 to 36 μm) or 16% to 17% of total neck length, female genital system monodelphic–opisthodelphic, anterior branch reduced to a uterine sac (26–29 μm) or 1.1 to 1.3 times the body diameter, long posterior uterus (25–28 μm) or 1.1 to 1.3 times the body diameter, V = 40 to 47, cylindroid female tail (17 to 24 μm, c = 31 to 38, c’ = 1.1 to 1.4), and males unknown. This taxon is easily distinguishable from other Margollus species by its smaller general size and more posterior vulva. A compendium of Margollus species is also presented.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.