Differentiation between dynamic instability and end-to-end annealing models for length changes of steady-state microtubules

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.     

A dynamic population of excitable cells: the taste receptor cells

Taste receptor cells (TRCs) represent an unique opportunity to study a dynamic population of excitable cells that undergoes two basic neurobiological processes: postnatal development and cell turnover. We have begun to investigate the functional properties of TRCs and how they mature over time by applying the patch-clamp technique to single cell in taste buds isolated from mouse vallate papilla during postnatal development. We have focussed our attention on a well-defined functional group of taste cells, called Na/OUT cells, and on their voltage-gated K+, and Cl- currents (I(K) and I(Cl), respectively). As in neurons, I(K) and I(Cl) underlie action potential waveform and firing properties in these cells. By analyzing the relative occurrence of I(K) and I(Cl) among cells, we found that in adult mice three different electrophysiological phenotypes of Na/OUT cells could be detected: cells with only I(K) (K cells); cells with both I(K) and I(Cl) (K + Cl cells); and cells with I(Cl) (Cl cells). On the contrary, at early developmental stages (2-4 postnatal day, PD) there were no Cl cells, which appeared at PD 8. The analysis of the changes in current amplitude (which continuously increased in developing cells) during postnatal development suggested that Cl cells and K + Cl cells likely represented a single functional line different from K cells. In addition, electrophysiological data were consistent with the interpretation that Cl cells derived from some K + Cl cells by suppression of I(K). The dynamics of the expression of I(K) and I(Cl) during postnatal development likely reflects a mechanism that could also operate during turnover.     

Opposite-end behaviour of dynamic microtubules

Microtubules are dynamic polar structures with different kinetic properties at the two ends. The inherent asymmetry of the microtubule lattice determines that the relationship between the addition reaction of tubulin-GTP and the associated hydrolysis of a tubulin-GTP on the polymer is different at the two ends of the microtubule. We present a unified treatment for both ends of the microtubule, using the principles of the Lateral Cap formulation for microtubule dynamic instability. This shows that the two ends can exhibit significantly different dynamic properties in terms of amplitudes and lifetimes of growth and shrinking, depending on the relative importance of longitudinal and lateral contacts in the coupling of tubulin-GTP hydrolysis. These predictions are readily amenable to experimental verification. This modelling suggests that fine details of the subunit-subunit interactions at the microtubule end can determine the characteristic differences in kinetic behaviour of the opposite ends of dynamic microtubules. Variation of these interactions would provide a potentially sensitive general mechanism for the control of such dynamics, both in vitro and in vivo.     

The length and disposition of cortical microtubules in plant cells fixed in glutaraldehyde—Osmium tetroxide

Serial sectioning has been used to show that the majority of circumferential microtubules lying in the cortex of root tip cells are much shorter than the cell circumference. The significance of this observation is briefly discussed.     

mDia mediates Rho-regulated formation and orientation of stable microtubules

Rho-GTPase stabilizes microtubules that are oriented towards the leading edge in serum-starved 3T3 fibroblasts through an unknown mechanism. We used a Rho-effector domain screen to identify mDia as a downstream Rho effector involved in microtubule stabilization. Constitutively active mDia or activation of endogenous mDia with the mDia-autoinhibitory domain stimulated the formation of stable microtubules that were capped and oriented towards the wound edge. mDia co-localized with stable microtubules when overexpressed and associated with microtubules in vitro. Rho kinase was not necessary for the formation of stable microtubules. Our results show that mDia is sufficient to generate and orient stable microtubules, and indicate that Dia-related formins are part of a conserved pathway that regulates the dynamics of microtubule ends.     

Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors

Homeostatic plasticity may compensate for Hebbian forms of synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), by scaling neuronal output without changing the relative strength of individual synapses. This delicate balance between neuronal output and distributed synaptic weight may be necessary for maintaining efficient encoding of information across neuronal networks. Here, we demonstrate that Arc/Arg3.1, an immediate-early gene (IEG) that is rapidly induced by neuronal activity associated with information encoding in the brain, mediates homeostatic synaptic scaling of AMPA type glutamate receptors (AMPARs) via its ability to activate a novel and selective AMPAR endocytic pathway. High levels of Arc/Arg3.1 block the homeostatic increases in AMPAR function induced by chronic neuronal inactivity. Conversely, loss of Arc/Arg3.1 results in increased AMPAR function and abolishes homeostatic scaling of AMPARs. These observations, together with evidence that Arc/Arg3.1 is required for memory consolidation, reveal the importance of Arc/Arg3.1's dynamic expression as it exerts continuous and precise control over synaptic strength and cellular excitability.     

Decline in excision circles requires homeostatic renewal or homeostatic death of naive T cells

When the TCR is formed in the thymus, fragments of DNA are excised from the T cell progenitor chromosome. These TCR rearrangement excision circles (TRECs) are stable, are not replicated in cell division and are therefore most frequent in naive T cells that have recently left the thymus. During life, the average TREC content of peripheral naive T cells decreases between one and two orders of magnitude in humans. It is generally believed that the age-dependent decrease in the production of naive T cells by the thymus is sufficient to explain the decrease in the TREC content. Here, we demonstrate that this decrease in thymic production is required, but it is not sufficient to explain the TREC data. Only if the decrease in thymic output is compensated by homeostasis can one explain the decrease in the TREC content. The homeostatic response can take two forms: when the total number of naive T cells declines, there could be an increase in the renewal rate or an increase of the average cellular lifespan.     

Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control.

A central question in cell biology is how cells determine the size of their organelles. Flagellar length control is a convenient system for studying organelle size regulation. Mechanistic models proposed for flagellar length regulation have been constrained by the assumption that flagella are static structures once they are assembled. However, recent work has shown that flagella are dynamic and are constantly turning over. We have determined that this turnover occurs at the flagellar tips, and that the assembly portion of the turnover is mediated by intraflagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the flagellar tips and causes the flagella to resorb. These results lead to a simple steady-state model for flagellar length regulation by which a balance of assembly and disassembly can effectively regulate flagellar length.     

Cortical control of plant microtubules

The cortical microtubule array of plant cells appears in early G(1) and remodels during the progression of the cell cycle and differentiation, and in response to various stimuli. Recent studies suggest that cortical microtubules are mostly formed on pre-existing microtubules and, after detachment from the initial nucleation sites, actively interact with each other to attain distinct distribution patterns. The plus end of growing microtubules is thought to accumulate protein complexes that regulate both microtubule dynamics and interactions with cortical targets. The ROP family of small GTPases and the mitogen-activated protein kinase pathways have emerged as key players that mediate the cortical control of plant microtubules.     

Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

Membrane nanotubes: dynamic long-distance connections between animal cells

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.     

Fgfs control homeostatic regeneration in adult zebrafish fins

Adult teleost fish and urodele amphibians possess a spectacular ability to regenerate amputated appendages, based on formation and maintenance of progenitor tissue called a blastema. Although injury-induced, or facultative, appendage regeneration has been studied extensively, the extent to which homeostatic regeneration maintains these structures has not been examined. Here, we found that transgenic inhibition of Fgf receptors in uninjured zebrafish caused severe atrophy of all fin types within 2 months, revealing a requirement for Fgfs to preserve dermal bone, joint structures and supporting tissues. Appendage maintenance involved low-level expression of markers of blastema-based regeneration, focused in distal structures displaying recurrent cell death and proliferation. Conditional mutations in the ligand Fgf20a and the kinase Mps1, factors crucial for regeneration of amputated fins, also caused rapid, progressive loss of fin structures in otherwise uninjured animals. Our experiments reveal that the facultative machinery that regenerates amputated teleost fins also has a surprisingly vigorous role in homeostatic regeneration.