Microtubules In Fibroblasts Research Articles

PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules.

Inhibition of the phospholipid phosphatase and tumor suppressor PTEN leads to excessive polarized cell growth during directed cell migration and neurite outgrowth. These processes require the precise regulation of both the actin and microtubule cytoskeleton. While PTEN is known to regulate actin dynamics through phospholipid modulation, whether and how PTEN regulates microtubule dynamics is unknown. Here, we show that depletion of PTEN leads to elevated levels of stable and post-translationally modified (detyrosinated) microtubules in fibroblasts and developing neurons. Further, PTEN depletion enhanced axon outgrowth, which was rescued by reducing the level of detyrosinated microtubules. These data demonstrate a novel role of PTEN in regulating the microtubule cytoskeleton. They further show a novel function of detyrosinated microtubules in axon outgrowth. Specifically, PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules. Our results suggest that PTEN’s role in preventing excessive cell growth in cancerous and neurodevelopmental phenotypes is partially exerted by stabilization and detyrosination of the microtubule cytoskeleton.

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration.

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells.

We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B-polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins-the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV-was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells.

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

Pure thoughts with impure proteins: permeabilized cell models of organelle motility.

Permeabilized cell models provide an experimental middle ground wherein the in vitro properties of mechanochemical proteins can be reconciled with the physical and topological constraints of the intact cell. Several well-studied examples of organelle motility are described here, including the actin-based cytoplasmic streaming of Characean algae, the microtubule-based aggregation and dispersion of pigment granules in chromatophores and the saltatory movements of vesicles along microtubules in fibroblasts and macrophages. The permeabilized models developed for these systems have helped to integrate observations in vivo with in vitro assays of motor proteins.

Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells.

In many cell types, a small subset of microtubules (MTs) are unusually long-lived compared with the majority of the MTs. These "stable" MTs may be important mediators of differentiative events since they are usually found aligned with developing asymmetries of cells undergoing morphogenesis. In addition to their longevity, the stable MTs are more resistant to drug depolymerization and are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). To determine the role of protein phosphorylation in the regulation of these stable MTs, we treated NIH 3T3 fibroblasts and TC-7 monkey kidney epithelial cells with okadaic acid (OA) and calyculin A, potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A), and then localized dynamic MTs and stable MTs with antibodies specific for tyrosinated tubulin (Tyrtubulin) and Glu-tubulin, respectively. OA at 0.1-10 microM caused a rapid and complete breakdown of Glu-MTs (MTs enriched in Glu-tubulin) in both cell types without substantially affecting the number of Tyr-MTs. While all concentrations of OA over this range resulted in a complete loss of Glu-MTs, the onset of Glu-MT breakdown was proportional to the logarithm of the OA concentration. The inactive analog of OA, 1-norokadaone, had no effect at any concentration. Calyculin A also caused a selective loss of Glu-MTs but was effective at 10 nM, consistent with its more potent inhibition of PP1. That the loss of Glu-MTs reflected the loss of stable MTs from the cells was shown by the absence of nocodazole-resistant MTs in OA-treated cells. OA did not appear to activate a MT-severing activity, since no MT fragments were observed after OA treatment of cells pretreated with taxol. These results suggest that PP1 and perhaps PP2A are involved in the regulation of MT stability in cells and show that the dynamic and stable subsets of MTs are regulated differentially by protein phosphorylation.

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific.

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.

New features of microtubule behaviour observed in vivo.

The microtubule cytoskeleton is thought to be intimately involved in generating and maintaining cell polarity and can generate many different morphological structures from a few structural elements. The mechanism by which these structures are generated has been partially elucidated from studies of microtubule polymerization both in vitro and in vivo. Microtubules in vitro exist in growing (polymerizing) and shrinking (depolymerizing) populations that interconvert infrequently. This behaviour, termed dynamic instability, permits microtubules in the cell rapidly to explore different arrangements and allows selective stabilization of specific morphologies. To investigate the regulation of these processes, we have implemented techniques for direct observation of fluorescently labelled microtubules and developed them to observe the dynamic behaviour of individual microtubules in single living cells. Sammak and Borisy recently used this technique to show that the dynamics of microtubules in fibroblasts is explained by dynamic instability. Although we also conclude here that dynamic instability explains much of microtubule behaviour in vivo, we find significant deviations from the properties of tubulin in vitro. These results suggest that local cytoplasmic factors strongly influence microtubule dynamics; such control has important implications for cellular morphogenesis.

Diminished microtubules in fibroblast cells derived from inherited dystrophic muscle explants.

MUSCULAR DYSTROPHY of the Duchenne type is inherited in a recessive, sex-linked fashion. The aetiology of the disease is still unclear but biochemical and morphological studies strongly suggest a cell surface membrane abnormality in Duchenne muscular dystrophy1–7. Even though there are artefacts due to surface condensation of water, freeze-fracture studies of skeletal muscle from patients with Duchenne dystrophy show a depletion of intramembrane particles in the protoplasmic and environmental faces of the muscle Plasma membrane8,9. In contrast, Ketelsen's freeze-fracture studies indicated that the number of particles in the inner membrane fracture face in muscular dystrophy increased two- or threefold beyond normal10. In other studies, it was demonstrated that patients and carriers of Duchenne muscular dystrophy exhibited diminished cap formation in their lymphocytes11,12. Although these experiments clearly support a membrane defect in muscular dystrophy, whether this is related to the aetiology of the disease is not yet clear. Several of these studies indicate that this membrane defect is an early and perhaps primary one in the aetiology of Duchenne's muscular dystrophy7. Even though the primary lesion in muscular dystrophy is usually thought to be myogenic or neurogenic the reported abnormalities in the lymphocyte11,12 and erythrocyte membrane13 indicate that the expression of muscular dystrophy may not necessarily be restricted to these tissues. There have been many studies on the dynamic interrelationships between cytoplasmic structures and the cell surface14,15 and models have been proposed which suggest that changes in the state of the cytoplasmic microfilaments and microtubules can alter the mobility and distribution of membrane surface receptors16. In an attempt to determine if the membrane defect in muscular dystrophy might be correlated with a cytoskeletal alteration, we have studied the distribution of cytoplasmic microfilaments (actin) and micro-tubules (tubulin) in fibroblasts derived from muscle expiants of both normal and dystrophic inbred chickens using indirect immunofluorescence methods with monospecific antibodies directed against actin and tubulin. We report here that fibroblasts from expiants of cardiac and skeletal muscle of dystrophic chickens show a dramatic reduction in the appearance of intact interphase microtubules compared with those from normal chickens.

Evidence for the involvement of microtubules in wound contraction

Many theories have been proposed for the mechanism of wound contraction, that phenomenon of wound closure in which the skin surrounding the tissue defect is drawn into the open wound. When agents that inhibit microtubular function, such as vinblastine and colchicine, were topically applied to actively contracting wounds, contraction stopped. Cytochalasin B, an agent that reportedly disrupts microfilaments, did not alter contraction. These results suggest that wound contraction is related to the functioning of microtubules in fibroblasts within the wound and is proceeding at its maximal rate. The results tend not to support the theory that the microfilament components of cells are involved in wound contraction.