Analysis of cellular mechanotransduction, the mechanism by which cells convert mechanical signals into biochemical responses, has focused on identification of critical mechanosensitive molecules and cellular components. Stretch-activated ion channels, caveolae, integrins, cadherins, growth factor receptors, myosin motors, cytoskeletal filaments, nuclei, extracellular matrix, and numerous other structures and signaling molecules have all been shown to contribute to the mechanotransduction response. However, little is known about how these different molecules function within the structural context of living cells, tissues, and organs to produce the orchestrated cellular behaviors required for mechanosensation, embryogenesis, and physiological control. Recent work from a wide range of fields reveals that organ, tissue, and cell anatomy are as important for mechanotransduction as individual mechanosensitive proteins and that our bodies use structural hierarchies (systems within systems) composed of interconnected networks that span from the macroscale to the nanoscale in order to focus stresses on specific mechanotransducer molecules. The presence of isometric tension (prestress) at all levels of these multiscale networks ensures that various molecular scale mechanochemical transduction mechanisms proceed simultaneously and produce a concerted response. Future research in this area will therefore require analysis, understanding, and modeling of tensionally integrated (tensegrity) systems of mechanochemical control.

/pdf/cellular-mechanotransduction-putting-all-the-pieces-together-46w4qt2qc2.pdf

Cellular mechanotransduction: putting all the pieces together again.

In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), extraction of actin-like or actomyosin-like proteins from cells other than muscle cells (54), and identification of activity resembling that of the actomyosin-adenosine triphosphatase system in a variety of nonmuscle tissues (40, 54) are consistent with the idea that portions of the complex, striated muscle contractile system may be present in more primitive contractile machinery. In the case of the egg cortex, calcium-activated contractions can be inhibited by cytochalasin. If, as seems likely, microfilaments are the agents activated by calcium, then it will be clear that they have the same calcium requirement as muscle. Biochemical analyses of primitive contractile systems are difficult to interpret. Ishikawa's important observation (31), that heavy meromyosin complexes with fine filaments oriented parallel to the surface of chondrocytes and perpendicular to the surface of intestinal epithelial cells, implies that both types of filaments are "actin-like" in this one respect. Yet, it is very likely that these actin-like filaments correspond respectively to the cytochalasin-insensitive sheath of glial and heart fibroblasts and the core filaments of oviduct microvilli. No evidence from our studies links contractility directly to these meromyosin-binding filaments. Apart from this problem, activity resembling that of the myosin-adenosine triphosphatase has been associated with the microtubule systems of sperm tails and cilia (55), but those organelles are insensitive to cytochalasin in structure and function. Clearly, a means must be found to distinguish between enzymatic activities associated with microfilament networks, microfilament bundles, microtubules, and the sheath filaments of migratory cells. Until such distinctions are possible, little of substance can be said about the molecular bases of primitive contractile systems. Three variables are important for the control of cellular processes dependent upon microfilaments: (i) which cells of a population shall manufacture and assemble the filaments; (ii) where filaments shall be assembled in cells; and (iii) when contractility shall occur. With respect to distribution among cells, the networks involved in cell locomotion are presumed to be present in all cells that have the potential to move in cell culture. In this respect, the networks can be regarded as a common cellular organelle in the sense that cytoplasmic microtubules are so regarded. In some developing systems, all cells of an epithelium possess microfilament bundles (7, 13), whereas, in others, only discrete subpopulations possess the bundles (5, 6). In these cases the filaments can be regarded as being differentiation products associated only with certain cell types. These considerations may be related to the fact that microfilament networks are associated with behavior of individual cells (such as migration, wound healing, and cytokinesis), whereas the bundles are present in cells that participate in coordinated changes in shape of cell populations. With respect to placement in cells, two alternatives are apparent, namely, localized or ubiquitous association with the plasma membrane. Microfilament bundles of epithelial cells are only found extending across the luminal and basal ends of cells. In this respect they contrast with desmosomal tonofilaments and with microtubules, each of which can curve in a variety of directions through the cell. The strict localization of microfilament bundles probably rests upon their association with special junctional complex insertion regions that are only located near the ends of cells. In the case of mitotically active cells, the orientation of the spindle apparatus may determine the site at which the contractile ring of microfilaments will form (4, 56); this raises the question of what sorts of cytoplasmic factors can influence the process of association between filament systems and plasma membranes. In contrast to such cases of localized distribution, contractile networks responsible for cell locomotion are probably found beneath all of the plasma membrane, just as the network of thrombosthenin may extend to all portions of the periphery of a blood platelet. This ubiquitous distribution probably accounts for the ability of a fibroblast or glial cell to establish an undulating membrane at any point on its edge, or of an axon to form lateral microspikes along its length. The third crucial aspect of control of these contractile apparatuses involves the choice of when contraction shall occur (and as a corollary the degree or strength of contraction that will occur). In the simplest situation, contraction would follow automatically upon assembly of the microfilament bundles or networks. In cleavage furrows of marine embryos (4), for instance, microfilaments are seen beneath the central cleavage furrow and at its ends, but not beyond, under the portion of plasma membrane that will subsequently become part of the furrow. This implies that the furrow forms very soon after the contractile filaments are assembled in the egg cortex. In other cases, microfilaments are apparently assembled but not in a state of (maximal?) contraction. Thus, networks are seen along the sides of migratory cells, although such regions are not then active as undulating membrane organelles. Similarly, microfilament bundles occur in all epithelial cells of the salivary gland (13), or pancreatic anlage (7), although only the ones at discrete points are thought to generate morphogenetic tissue movements. Likewise, bundles begin to appear as early as 12 hours after estrogen administration to oviduct, although visible tubular gland formation does not start until 24 to 30 hours. Finally, streaming in plant cells can wax and wane, depending upon external factors such as auxin (57). All of these cases imply a control mechanism other than mere assembly of the microfilament systems and even raise the possibility that within one cell some filaments may be contracting while others are not. In discussing this problem, it must be emphasized that different degrees of contraction or relaxation cannot as yet be recognized with the electron microscope. In fact, every one of the cases cited above could be explained by contraction following immediately upon some subtle sort of "assembly." Inclusive in the latter term are relations between individual filaments, relations of the filaments and their insertion points on plasma membrane, and quantitative alterations in filament systems. Furthermore, the critical role of calcium and high-energy compounds in muscle contraction suggest that equivalent factors may be part of primitive, cytochalasinsensitive systems. The finding that calcium-induced contraction in the cortex of eggs is sensitive to cytochalasin strengthens that supposition and emphasizes the importance of compartmentalization of cofactors as a means of controlling microfilaments in cells.

https://science.sciencemag.org/content/sci/171/3967/135.full.pdf

Microfilaments in Cellular and Developmental Processes

We review recent advances in laser cell surgery, and investigate the working mechanisms of femtosecond laser nanoprocessing in biomaterials with oscillator pulses of 80-MHz repetition rate and with amplified pulses of 1-kHz repetition rate. Plasma formation in water, the evolution of the temperature distribution, thermoelastic stress generation, and stress-induced bubble formation are numerically simulated for NA=1.3, and the outcome is compared to experimental results. Mechanisms and the spatial resolution of femtosecond laser surgery are then compared to the features of continuous-wave (cw) microbeams. We find that free electrons are produced in a fairly large irradiance range below the optical breakdown threshold, with a deterministic relationship between free-electron density and irradiance. This provides a large ‘tuning range’ for the creation of spatially extremely confined chemical, thermal, and mechanical effects via free-electron generation. Dissection at 80-MHz repetition rate is performed in the low-density plasma regime at pulse energies well below the optical breakdown threshold and only slightly higher than used for nonlinear imaging. It is mediated by free-electron-induced chemical decomposition (bond breaking) in conjunction with multiphoton-induced chemistry, and hardly related to heating or thermoelastic stresses. When the energy is raised, accumulative heating occurs and long-lasting bubbles are produced by tissue dissociation into volatile fragments, which is usually unwanted. By contrast, dissection at 1-kHz repetition rate is performed using more than 10-fold larger pulse energies and relies on thermoelastically induced formation of minute transient cavities with lifetimes <100 ns. Both modes of femtosecond laser nanoprocessing can achieve a 2–3 fold better precision than cell surgery using cw irradiation, and enable manipulation at arbitrary locations.

Mechanisms of femtosecond laser nanosurgery of cells and tissues

In 1993, a Commentary in this journal described how a simple mechanical model of cell structure based on tensegrity architecture can help to explain how cell shape, movement and cytoskeletal mechanics are controlled, as well as how cells sense and respond to mechanical forces (J. Cell Sci. 104, 613-627). The cellular tensegrity model can now be revisited and placed in context of new advances in our understanding of cell structure, biological networks and mechanoregulation that have been made over the past decade. Recent work provides strong evidence to support the use of tensegrity by cells, and mathematical formulations of the model predict many aspects of cell behavior. In addition, development of the tensegrity theory and its translation into mathematical terms are beginning to allow us to define the relationship between mechanics and biochemistry at the molecular level and to attack the larger problem of biological complexity. Part I of this two-part article covers the evidence for cellular tensegrity at the molecular level and describes how this building system may provide a structural basis for the hierarchical organization of living systems--from molecule to organism. Part II, which focuses on how these structural networks influence information processing networks, appears in the next issue.

/pdf/tensegrity-i-cell-structure-and-hierarchical-systems-biology-3c0xcl0gcm.pdf

Tensegrity I. Cell structure and hierarchical systems biology.

Amyloid Precursor Protein Trafficking, Processing, and Function

1. A radioautographic study of the cells of the root tips of wheat incubated with d-[1- or 6-3H]glucose has shown that labelled material is formed in the golgi apparatus of the root-cap cells. This material passed to the vesicles associated with the golgi bodies and then moved through the cytoplasm across the plasmalemma and was incorporated into the cell wall and slime layer of the tissue. 2. Analysis of the labelled material extracted from the root tips showed that the bulk of the radioactive material was polysaccharide; there were relatively small amounts of labelled lipids and protein in the tissue. 3. Starch was formed from the exogenous labelled glucose and it was located in the plastids of the cell. The synthesis of starch depended on the metabolic activity of the cells, which varied with the position of the cell in the various tissues of the root tip and with the amount of the exogenous glucose. 4. Isolation of the radioactive polysaccharides from the root tip incubated in the radioactive glucose has shown that the glucose was very rapidly incorporated into the galactosyl residues of the polymers. 5. Analysis of the radioactive polysaccharides has indicated that the material transported in the golgi vesicles is probably pectic substance. 6. A scheme for the synthesis of the storage and wall polysaccharides by separate routes and their location within the cell has been put forward.

/pdf/a-function-of-the-golgi-apparatus-in-polysaccharide-2chb5y63rv.pdf

A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat.

The fine-structural changes accompanying mitosis in meristematic cells of the roots and coleoptile tissue of wheat have been studied. A band of microtubules encircling the nucleus appeared in the cytoplasm before the cells entered prophase. These microtubules were oriented at right angles to the direction of the mitotic spindle and were located at the position on the mother cell wall where the future cell plate dividing the daughter cells would have joined it. During prophase the number of microtubules in this preprophase band decreased and eventually disappeared, while microtubules were found to be aligned along the spindle axis. These spindle microtubules appeared as a cone-shaped array of units radiating from the polar zones of the spindle and passing very close tangentially to the nucleus. At late prophase they penetrated the disintegrating nuclear envelope and were seen between the chromosomes. During metaphase and anaphase many microtubules were present running throughout the length of the spindle, and others were found to be attached to chromosomes. Paired sister chromosomes were found joined to microtubules from opposite poles of the spindle. The position and orientation of the lamellae of the endoplasmic reticulum which invaded the spindle from the two poles was closely related to the position and alignment of the microtubules. During the formation of the cell plate vesicles were seen to be collected between the microtubules. As the vesicles fused to form the plate the microtubules were found only at its growing edge, where the vesicles were still being aligned. At the initial stage of its formation the microtubules passed right through the plate, but as it extended they appeared to end at the plate region. The results of the investigation are discussed in relation to the descriptions of mitosis and cytokinesis based on optical microscopy of living cells.

/pdf/organization-of-microtubules-and-endoplasmic-reticulum-dujlkgwxkl.pdf

Organization of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems.

Quantitative methods and associated kinetic analyses have been used for the first time to study detailed aspects of the settlement and adhesion of various types of Enteromorpha popagule. Time course experiments showed that quadri and biflagellate zoospores and zygotes adhered rapidly, but a proportion within any one population appeared to be incompetent at adhering to the substratum. Kinetic (Scatchard) analysis of adhesion experiments performed at a range of zoospore concentrations revealed density-dependent effects not previously reported, with positive cooperativity at low spore densities and negative cooperativity at high spore densities. High-resolution video microscopy was used for the first time to reveal details of the various stages in the settlement and adhesion of zoospores and zygotes. Novel observations were made of an initial, temporary phase of attachment via the apical papilla, followed by a permanent phase of commitment, characterized by discharge of adhesive-containing cytoplasmic vesicles, as the cell contracted against the surface, and adsorption of flagella. The phase of commitment was followed by exploitation of the surface through amoeboid-like movements at the interface. Gregarious settlement behavior was frequently observed leading to the formation of rafts of cells. The possible mechanisms and significance of density-dependent spore adhesion are discussed.

Primary adhesion of enteromorpha (chlorophyta, ulvales) propagules: quantitative settlement studies and video microscopy1

The effects of colchicine on the ultrastructure of dividing plant cells, xylem wall differentiation and distribution of cytoplasmic microtubules

During the formation of stomata in the young leaves of wheat the cells divide in a characteristic manner; two of the cell divisions are asymmetrical and produce cells of unequal sizes. A study of the fine structure of the cells during mitosis has shown that a band of microtubules appears at each preprophase stage. This band, although it is not present in the subsequent stages of mitosis, indicates the location on the wall of the mother cell where the cell plate will join it at the final division of the cytoplasm at telophase. Thus the future plane of cell division is indicated by these microtubules at preprophase. Microtubules are also found at the growing edge of the cell plate and appear to function in directing the vesicles which are brought up to extend the plate. The cell plate which is formed to cut off the subsidiary cells on either side of the guard mother cell is curved, and the microtubules present in conjunction with this plate during its formation could function to align and hold it on the required position. The relationship of the guard mother cell to the divisions of the adjacent epidermal cells which form the subsidiary cells is discussed, and related to general problems of growth and differentiation.

/pdf/cell-division-in-the-formation-of-the-stomatal-complex-of-4iu582odn1.pdf

Jeremy D. Pickett-Heaps

Papers

A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat.

Organization of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems.

Primary adhesion of enteromorpha (chlorophyta, ulvales) propagules: quantitative settlement studies and video microscopy1

The effects of colchicine on the ultrastructure of dividing plant cells, xylem wall differentiation and distribution of cytoplasmic microtubules

Cell division in the formation of the stomatal complex of the young leaves of wheat.