Intrusive growth of flax phloem fibers is of intercalary type

Flax (Linum usitatissimum L.) phloem fibers elongate considerably during their development and intrude between existing cells. We questioned whether fiber elongation is caused by cell tip growth or intercalary growth. Cells with tip growth are characterized by having two specific zones of cytoplasm in the cell tip, one with vesicles and no large organelles at the very tip and one with various organelles amongst others longitudinally arranged cortical microtubules in the subapex. Such zones were not observed in elongating flax fibers. Instead, organelles moved into the very tip region, and cortical microtubules showed transversal and helical configurations as known for cells growing in intercalary way. In addition, pulse-chase experiments with Calcofluor White resulted in a spotted fluorescence in the cell wall all over the length of the fiber. Therefore, it is concluded that fiber elongation is not achieved by tip growth but by intercalary growth. The intrusively growing fiber is a coenocytic cell that has no plasmodesmata, making the fibers a symplastically isolated domain within the stem. Electronic Supplementary Material Supplementary material is available for this article at     

Cotton fibers can undergo cell division

Ovular culture was used to determine the cell cycle aspects of cotton fiber cells. Each ovule (Gossypium hirsutum, cultivar, MD51 ne) grown under the conditions used has ~10 000 fiber cells at 4 d postanthesis. About 25% of these cells divide when ovules are cultured at 34C. Mitosis occurs after fiber cells differentiate, producing multicelled fibers. The basal and tip cells of multicelled fibers have the same characteristics as the polar ends of single-celled fibers. Most cell division occurs in ovules cultured at 2-3 d postanthesis. Multicelled fibers are rare in ovules cultured at 1 d postanthesis and absent if cultured at 7 d postanthesis. No multicelled fibers are detectable on ovules sampled from the plant regardless of age. Fiber cell division occurs in the absence of exogenous hormones. The addition of IAA and GA3 to the medium lowers the frequency of multicelled fibers. IAA alone further reduces their frequency, while GA3 by itself has no effect. The number of fiber cells per cultured ovule ranges between 9462 and 11 087 and is not significantly different from the 9892 seen in the plant at 4 d postanthesis. These findings show that a subpopulation of fiber cells, fully differentiated in appearance, retain cell cycle functions up to 4 d postanthesis.     

How cotton fibers elongate: a tale of linear cell-growth mode

Cotton fibers (cotton lint) are single-celled trichomes that differentiate from the ovule epidermis. Unidirectional and fast-growing cells generally expand at the dome-shaped apical zone (tip-growth mode); however, previous studies suggest that elongating fiber cells expand via a diffuse-growth mode. Tip-localized Ca(2+) gradient and active secretary vesicle trafficking are two important phenomena of tip-growth. Recently, a high Ca(2+) gradient is found in the cytoplasm of fast-elongating cotton fiber cells near the growing tip. Several protein coding genes participating in vesicle coating and transport are highly expressed in elongating fiber cells. Taken together with the observation that ethylene acts as a positive regulator for cotton fiber and several Arabidopsis tissues that are known to elongate via tip growth prompted us to propose a linear-growth mode for similar cell types.     

A multi-modular tensegrity model of an actin stress fiber

Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on the extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model can also explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors and represent a new handle on multi-scale modeling of living materials.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

Pax7 reveals a greater frequency and concentration of satellite cells at the ends of growing skeletal muscle fibers.

The main sites of longitudinal growth in skeletal muscle are the ends of the fibers. This study tests the hypothesis that satellite cells (SCs) are at a greater frequency (#SC nuclei/all nuclei within basal laminae) and concentration (closer together) within growing fiber ends of posthatch chicken pectoralis. SCs were localized by their Pax7 expression, and fiber ends were identified by their retention of neonatal myosin heavy chains and small cross-sectional profiles. Whereas SC frequency decreased from about 20% at 9 days posthatch to <5% at 115 days, fiber ends retained a frequency of approximately 16%. Calculated mean area of sarcolemma per SC revealed higher concentrations of SCs at fiber ends. There was also a strong inverse correlation between SC frequency and fiber profile cross-sectional size throughout development. This study suggests that SCs at fiber ends play a key role in the longitudinal growth of muscle fibers, and that fiber profile size may impact SC distribution.     

Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.     

Granule cell migration in developing rat cerebellum. Influence of neonatal hypo- and hyperthyroidism.

The rate of cerebellar granule cell migration is altered by neonatal hypo- and hyperthyroidism in a manner similar to previously reported effects on the growth of granule cell axons, the parallel fibers, suggesting that the two processes may be intimately linked. Altered rates of granule cell acquisition in these experimental animals reflect changes in germinal cell proliferation in the external granular layer (EGL), movement of postmitotic cells within the EGL, as well as the rate and time course of granule cell migration. Results of this study support the hypothesis that granule cells migrate to the internal granular layer by translocation of the cell body through the descending portion of the growing parallel fiber, rather than by amoeboid-like migration of the perikaryon trailing the elongating parallel fiber behind.     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.     

Development of gelatinous (reaction) fibers in stems of Gnetum gnemon (Gnetales)

In extraxylary tissues of the stem Gnetum gnemon produces gelatinous fibers that can also function as reaction or tension fibers. These gelatinous fibers occur in all axes in the outer cortex and in displaced axes progressively in the middle and inner cortex and finally in the secondary phloem. Early cell differentiation in the cortex produces initials of laticifers that are unique in gymnosperms. Subsequently narrow fibers differentiate from cells that undergo both extensive passive elongation, as a result of internodal elongation, together with their active apical intrusive growth. Outer fibers always complete secondary wall development and become an important mechanical component of stems. Differentiation of fiber initials continues in the middle and inner cortex, but secondary wall formation can only be determined by a gravimorphic stimulus that produces eccentric development of fibers. Further eccentric development of fibers then continues in the outer secondary phloem from dedifferentiated phloem parenchyma cells that initially undergo extensive intrusive growth. All such cells have characteristic features of tension fibers of angiosperms. They exhibit a pronounced purely cellulosic innermost layer of the secondary wall (Sg layer). In addition, fiber initials are coenocytic, including up to eight nuclei that become distributed uniformly throughout the length of the cell. Mature macerated fibers are markedly brittle, making accurate length measurements difficult. Although cytologically uniform, these fibers thus originate from two kinds of initial (primary and secondary). They also differ in their response to a gravimorphic stimulus determined by their times of inception and their eccentric location. These cells show a suite of positional and gravimorphic responses that illustrate the complexity of plant cell differentiation.     

Possible translocation of actin and alpha-actinin along stress fibers

We have employed fluorescent analogue cytochemistry and fluorescence photobleaching to study the mobility of actin and alpha-actin along stress fibers. Rhodamine-labeled actin or alpha-actinin microinjected into embryonic chick cardiac fibroblasts soon became incorporated into stress fibers. A pulse of a laser microbeam was used to photobleach small spots on the fluorescent stress fibers. Images of the bleached fiber were recorded with an intensified image processing system at 2-3 min intervals. The distance between the bleached spot and the terminus of the stress fiber, which remained stationary throughout the experiment, was then measured in the successive images. Movement of bleached spots was detected along stress fibers located in the apparently trailing processes of polygonal fibroblasts, and only occurred in one direction: away from the distal tip of the stress fiber. The rate of movement calculated for alpha-actinin-injected cells was 0.24 +/- 0.12 micron/min, for actin-injected cells, 0.29 +/- 0.11 micron/min. The rate did not seem to be affected by the location of the spot relative to the distal end of the stress fiber unless the spot was located within the most distal 5 microns of the stress fiber. Anti-myosin antibody staining indicated that stress fibers which demonstrated translocation were relatively depleted of myosin. The apparent translocation of proteins along stress fibers, possibly generated by stretching, may be related to the retraction of cell processes during locomotion.     

A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.     

Senescence and apoptosis of protoplasts from flax fibers: an ultrastructural analysis

Plant fibers represent specialized cells that perform a mechanical function. Their development includes the following phases, typical for the most plant cells: anlage, extension growth, specialization, senescence, and apoptosis. Ultrastructural analysis of these cells has been carried out at the late phases of their development (senescence and apoptosis) using flax phloem fibers, a classical object for the analysis of sclerenchyma fiber formation. The results of the performed analysis show that flax fiber protoplasts remain viable until the end ofa vegetation season. The ultrastructural analysis of flax phloem fibers has not revealed any typical apoptosis manifestations. Gradual degradation of the cytoplasm starts during the active thickening of a secondary cell wall and manifests via the intensification of autolytic processes, causing a partial loss of cell content. The final stage represents the breaking of tonoplast integrity. The obtained data allow us to suppose that the apoptosis of flax fibers occurs during their senescence, and its program is similar to the cell death program realized in the xylem fibers of woody plants.     

Two types of very long visual fibers found in the optic lobe of the flesh-fly,Boettcherisca peregrina

Summary Horseradish peroxidase (HRP) uptake (through a Corneal incision) in photoreceptor cells of the compound eye of Boettcherisca peregrina, resulted in the labeling of two types of very long visual fibers. One of them (the long fiber, If) penetrates through the lamina and medulla, and directly terminates within the lobula. The other (the bypass fiber, bpf) terminates in the medulla, like the axons of R7 and R8 photoreceptor cells, but the fibers run a considerably roundabout course. The photoreceptor cells with these very long fibers are region-dependent within the retina. Both the If and bpf are found almost exclusively in the male fly.     

Processes of protoplast senescence and death in flax fibers: An ultrastructural analysis

Plant fibers represent specialized cells that perform a mechanical function. Their development includes the following phases, typical for the most plant cells: determination, extension growth, specialization, senescence, and death. Ultrastructural analysis of these cells has been carried out at the late phases of their development (senescence and dying off) using flax phloem fibers, a classical object for the analysis of sclerenchyma fiber formation. The results of the performed analysis show that flax fiber protoplasts remain viable until the end of a vegetation season. The ultrastructural analysis of flax phloem fibers has not revealed any typical apoptosis features. Gradual degradation of the cytoplasm starts during the active thickening of a secondary cell wall and occurs via the intensification of autolytic processes, causing a partial loss of cell content. The rupture of tonoplast is the final stage. The obtained data allow us to suppose that the protoplast dying off occurs during process of the senescence, and its program is similar to the cell death program realized in the xylem fibers of woody plants.     

Nitrogen deposition and its contribution to nitrogen cycling and associated soil processes

TIP1 is a gene defined by an X-ray induced allele tip1–2 and a previously described EMS-induced allele tip1−1 . TIP1 is involved in plant cell growth. tip1–2 plants display growth defects throughout the plant and exhibit defects in both root-hair and pollen-tube growth. tip1–2 plants are partly male sterile resulting from a combination of pollen germination and pollen-tube defects; their root-hairs are short, exhibit a tendency to branch and 2–4 hairs can initiate from each hair cell. They are also slightly dwarf in stature as a result of a general decrease in cell growth indicating that TIP1 activity is required for general cell growth. We propose a role for TIP in both the initiation and maintenance of growth in tip-growing cells. In addition TIP1 activity is required for normal cell expansion (non-tip cell growth) indicating that TIP1 is not exclusively involved in tip-growth.     

Satellite cells from dystrophic (mdx) mouse muscle are stimulated by fibroblast growth factor in vitro

Satellite cells cultured from dystrophic (mdx) and from control mouse hindlimb muscles grow and fuse to form muscle fibers within 4-5 days. Total cell number and muscle-fiber formation are stimulated by bovine fibroblast growth factor (FGF). At low FGF levels (0.02-0.20 ng/ml) control satellite cells as well as fibroblasts are unresponsive, while mdx satellite cells show three- to four-fold increases in growth. Control cells do not begin to respond until FGF levels reach 1-5 ng/ml. Heparin, a major constituent of muscle fiber basal lamina, inhibits myogenesis in these mouse muscle cultures. The heightened sensitivity of mdx satellite cells to FGF may permit high rates of new fiber formation in vivo without a parallel hyperplasia in the muscle fibroblast population. This finding may be important in explaining successful regeneration in mdx muscle in vivo and the fact that mdx animals escape the catastrophic symptoms seen in the related human Duchenne muscular dystrophy.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Merkel cell-nerve cell interaction undergoes formation of a synapse-like structure in a primary culture

Merkel cells have been assumed to guide nerve fibers to the skin. However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable established culture system of Merkel cells. Here we show that Merkel cells isolated from rat footpad skin were successfully cultured in a monolayer with keratinocytes. Keratinocytes did not affect any structural changes in Merkel cells. When nerve cells (NG108-15 or PC12) were added to the culture system, both nerve fibers and cytoplasmic processes of Merkel cells outgrew and cooperatively organized synapse-like structures at their contact points. Nerve cells promoted Merkel cell survival, compared with keratinocytes only. Merkel cell proliferation was not detected in all conditions, even with nerve growth factor, neurotrophin-3, interleukin-6 and tumor necrosis factor-alpha. The data suggest, firstly, that Merkel cells may guide nerve fibers to the skin by interacting with nerve cells; and, secondly, that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells, although Merkel cells may be a terminally differentiated cell type. Our method could open a way to study Merkel cell biology.