RESUMO
The migration of tissue cells requires interplay between the microtubule and actin cytoskeletal systems. Recent reports suggest that interactions of microtubules with actin dynamics creates a polarization of microtubule assembly behavior in cells, such that microtubule growth occurs at the leading edge and microtubule shortening occurs at the cell body and rear. Microtubule growth and shortening may activate Rac1 and RhoA signaling, respectively, to control actin dynamics. Thus, an actin-dependent gradient in microtubule dynamic-instability parameters in cells may feed back through the activation of specific signalling pathways to perpetuate the polarized actin-assembly dynamics required for cell motility.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Microtúbulos/metabolismo , Transdução de Sinais/fisiologia , Retroalimentação , Humanos , Modelos BiológicosRESUMO
Microtubules are involved in actin-based protrusion at the leading-edge lamellipodia of migrating fibroblasts. Here we show that the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole activates Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. We suggest that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration.
Assuntos
Microtúbulos/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Células 3T3/efeitos dos fármacos , Células 3T3/fisiologia , Células 3T3/ultraestrutura , Actinas/metabolismo , Animais , Movimento Celular/fisiologia , Cinética , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Paclitaxel/farmacologiaRESUMO
We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Microtúbulos/fisiologia , Animais , Polaridade Celular , Células Cultivadas , Pulmão/citologia , Pulmão/fisiologia , Microinjeções , Microscopia de Fluorescência , Microscopia de Vídeo/métodos , Microtúbulos/ultraestrutura , Salamandridae , Fatores de Tempo , Tubulina (Proteína)/análiseRESUMO
We discovered by using high resolution video microscopy, that membranes become attached selectively to the growing plus ends of microtubules by membrane/microtubule tip attachment complexes (TACs) in interphase-arrested, undiluted, Xenopus egg extracts. Persistent plus end growth of stationary microtubules pushed the membranes into thin tubules and dragged them through the cytoplasm at the approximately 20 microns/min velocity typical of free plus ends. Membrane tubules also remained attached to plus ends when they switched to the shortening phase of dynamic instability at velocities typical of free ends, 50-60 microns/min. Over time, the membrane tubules contacted and fused with one another along their lengths, forming a polygonal network much like the distribution of ER in cells. Several components of the membrane networks formed by TACs were identified as ER by immunofluorescent staining using antibodies to ER-resident proteins. TAC motility was not inhibited by known inhibitors of microtubule motor activity, including 5 mM AMP-PNP, 250 microM orthovanadate, and ATP depletion. These results show that membrane/microtubule TACs enable polymerizing ends to push and depolymerizing ends to pull membranes into thin tubular extensions and networks at fast velocities.
Assuntos
Interfase/fisiologia , Membranas Intracelulares/fisiologia , Proteínas dos Microtúbulos/fisiologia , Óvulo/fisiologia , Animais , Feminino , Microscopia de Interferência , Proteínas dos Microtúbulos/antagonistas & inibidores , Microtúbulos/metabolismo , Polímeros/metabolismo , Ratos , Extratos de Tecidos/fisiologia , XenopusRESUMO
Although it is generally believed that microtubules have minus ends bound to the centrosome and free plus ends that exhibit dynamic instability, recent observations show that the minus ends can be free and that modulation of dynamic instability at both ends can result in treadmilling and flux in interphase cells.
Assuntos
Interfase/fisiologia , Microtúbulos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Movimento/fisiologiaRESUMO
BACKGROUND: The microtubule-dependent motility of endoplasmic reticulum (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have arisen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movement mechanism' in which ER attaches to moving microtubules; and the 'tip attachment complex (TAC) mechanism' in which ER tubules attach to growing plus ends of microtubules. RESULTS: We have used multi-wavelength time-lapse epifluorescence microscopy to image the dynamic interactions between microtubules (by microinjection of X-rhodamine-labeled tubulin) and ER (by DiOC6(3) staining) in living cells to determine which mechanism contributes to the formation and motility of ER tubules in migrating cells in vivo. Newly forming ER tubules extended only in a microtubule plus-end direction towards the cell periphery: 31.4% by TACs and 68.6% by the membrane sliding mechanism. ER tubules, statically attached to microtubules, moved towards the cell center with microtubules through actomyosin-based retrograde flow. TACs did not change microtubule growth and shortening velocities, but reduced transitions between these states. Treatment of cells with 100 nM nocodazole to inhibit plus-end microtubule dynamics demonstrated that TAC motility required microtubule assembly dynamics, whereas membrane sliding and retrograde-flow-driven ER motility did not. CONCLUSIONS: Both plus-end-directed membrane sliding and TAC mechanisms make significant contributions to the motility of ER towards the periphery of living cells, whereas ER removal from the lamella is powered by actomyosin-based retrograde flow of microtubules with ER attached as cargo. TACs in the ER modulate plus-end microtubule dynamics.
Assuntos
Retículo Endoplasmático/fisiologia , Membranas Intracelulares/fisiologia , Pulmão/fisiologia , Microtúbulos/fisiologia , Animais , Células Cultivadas , Retículo Endoplasmático/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Membranas Intracelulares/ultraestrutura , Pulmão/citologia , Microscopia de Fluorescência , Microscopia de Vídeo , Microtúbulos/ultraestrutura , Salamandridae , Suínos , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.
Assuntos
Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Linhagem Celular , Humanos , Processamento de Imagem Assistida por Computador , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Salamandridae , Suínos , Xenopus laevisRESUMO
To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.
Assuntos
Citoesqueleto/fisiologia , Junções Intercelulares/fisiologia , Pulmão/citologia , Mucosa Respiratória/fisiologia , Transativadores , Actinas/metabolismo , Animais , Adesão Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Salamandridae , alfa Catenina , beta CateninaRESUMO
The myofibrillar cytoskeleton of skeletal muscle is made up of two distinct sets of filaments, the exosarcomeric cytoskeleton and the endosarcomeric cytoskeleton. The exosarcomeric cytoskeleton consists of intermediate filaments (IF) composed of the proteins desmin, vimentin, and synemin. The IF are arranged both longitudinally and transversely around the fiber. The longitudinal filaments run from Z-disc to Z-disc, enveloping the myofibril in order to serve as attachment sites for mitochondria, nuclei, and the sarcolemma, as well as limiting the sarcomere's extensibility. The transverse filaments link adjacent myofibrils at the Z-disc and are responsible for the fibril's axial register, and thus the striated appearance of muscle. The endosarcomeric cytoskeleton acts as a third filament system that coexists with actin and myosin within the sarcomere. This system is believed to be extensible and is made up of the giant proteins, titin and nebulin. Titin is believed to be responsible for resting muscle elasticity, as well as the central position of myosin in the sarcomere. Nebulin's role is proposed to be the maintenance of actin's lattice array. Following various types of intense exercise, pathological changes in muscle morphology have been documented. These include Z-disc streaming, sarcomerogenesis, and decentralization of myosin filaments within the sarcomere. It is hypothesized that disruption of the transverse IF system may cause Z-disc streaming, whereas degradation of titin filaments may affect myosin's position in the sarcomere.
Assuntos
Citoesqueleto/ultraestrutura , Exercício Físico/fisiologia , Músculos/ultraestrutura , Humanos , Microscopia EletrônicaAssuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Vídeo/métodos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Animais , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais , Fluoresceínas , Microscopia de Fluorescência/métodos , Microscopia de Interferência/métodos , Movimento/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Fotoquímica , Salamandridae , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.
Assuntos
Actinas/química , Células Epiteliais/citologia , Microscopia de Fluorescência/métodos , Pseudópodes/química , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Algoritmos , Animais , Biofísica/métodos , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular , Movimento Celular , Células Cultivadas , Depsipeptídeos/química , Processamento de Imagem Assistida por Computador , Cinética , Microscopia Confocal , Modelos Moleculares , Modelos Estatísticos , Polímeros/química , Potoroidae , Pseudópodes/metabolismo , Tiazóis/química , Tiazolidinas , Fatores de TempoRESUMO
This unit describes an in vitro assay that uses video-enhanced differential interference contrast (VE-DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme-nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE-DIC microscopy.
Assuntos
Ensaios de Migração Celular/métodos , Movimento Celular/fisiologia , Microscopia de Interferência/métodos , Microscopia de Vídeo/métodos , Microtúbulos/ultraestrutura , Organelas/ultraestrutura , Animais , Ensaios de Migração Celular/instrumentação , Movimento Celular/efeitos dos fármacos , Cultura em Câmaras de Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Microscopia de Interferência/instrumentação , Microscopia de Vídeo/instrumentação , Microtúbulos/fisiologia , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/farmacologia , Organelas/efeitos dos fármacos , Organelas/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ouriços-do-Mar , Sus scrofaRESUMO
Migrating cells display a characteristic polarization of the actin cytoskeleton. Actin filaments polymerise in the protruding front of the cell whereas actin filament bundles contract in the cell body, which results in retraction of the cell's rear. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42. Although the microtubule cytoskeleton is also polarized in a migrating cell, and microtubules are essential for the directed migration of many cell types, their role in cell motility is not well understood at a molecular level. Here, we discuss the potential molecular mechanisms for interplay of microtubules, actin and Rho GTPase signalling in cell polarization and motility. Recent evidence suggests that microtubules locally modulate the activity of Rho GTPases and, conversely, Rho GTPases might be responsible for the initial polarization of the microtubule cytoskeleton. Thus, microtubules might be part of a positive feedback mechanism that maintains the stable polarization of a directionally migrating cell.
Assuntos
Movimento Celular/fisiologia , Microtúbulos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Citoesqueleto/metabolismo , Humanos , Microtúbulos/fisiologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Fluorescent speckle microscopy (FSM) is a technology for analysing the dynamics of macromolecular assemblies. Originally, the effect of random speckle formation was discovered with microtubules. Since then, the method has been expanded to other proteins of the cytoskeleton such as f-actin and microtubule binding proteins. Newly developed, specialized software for analysing speckle movement and photometric fluctuation in the context of polymer transport and turnover has turned FSM into a powerful method for the study of cytoskeletal dynamics in cell migration, division, morphogenesis and neuronal path finding. In all these settings, FSM serves as the quantitative readout to link molecular and genetic interventions to complete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering of molecular regulation of the cytoskeleton. Fully automated FSM assays can also be applied to live-cell screens for toxins, chemicals, drugs and genes that affect cytoskeletal dynamics. We envision that FSM has the potential to become a core tool in automated, cell-based molecular diagnostics in cases where variations in cytoskeletal dynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. In this paper, we review the origins of FSM, discuss these most recent technical developments and give a glimpse to future directions and potentials of FSM. It is written as a complement to the recent review (Waterman-Storer & Danuser, 2002, Curr. Biol., 12, R633-R640), in which we emphasized the use of FSM in cell biological applications. Here, we focus on the technical aspects of making FSM a quantitative method.
Assuntos
Citoesqueleto/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Polímeros/metabolismo , Actinas/metabolismo , Animais , Células Epiteliais/metabolismo , Pulmão/citologia , Óvulo/metabolismo , Salamandridae , XenopusRESUMO
p150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein. Cloning of a cDNA encoding p150Glued from rat revealed 31% amino acid sequence identity with the product of the Drosophila gene, Glued. A dominant Glued mutation results in neuronal disruption; null mutations are lethal. However, the Glued gene product has not been characterized. To determine whether the Glued polypeptide is functionally similar to vertebrate p150Glued, we characterized the Glued protein in the Drosophila S-2 cell line. Antibodies raised against Glued were used to demonstrate that this protein sediments exclusively at 20 S, and associates with microtubules in a salt- and ATP-dependent manner. Immunoprecipitations from S-2 cytosol with the anti-Glued antibody resulted in the co-precipitation of subunits of both cytoplasmic dynein and the dynactin complex. An affinity column with covalently bound Glued protein retained cytoplasmic dynein from S-2 cytosol. Based on these observations, we conclude that Glued is a component of a dynactin complex in Drosophila and binds to cytoplasmic dynein, and therefore the mutant Glued phenotypes can be interpreted as resulting from a disruption in the function of the dynactin complex.
Assuntos
Drosophila/genética , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Animais , Drosophila/metabolismo , Complexo Dinactina , Ratos , Alinhamento de Sequência , Análise de Sequência , Vertebrados/metabolismoRESUMO
p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.
Assuntos
Actinas/metabolismo , Dineínas/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Complexo Dinactina , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.
Assuntos
Microscopia de Fluorescência , Microtúbulos , Animais , Simulação por Computador , Microscopia de Fluorescência/métodos , SalamandridaeRESUMO
The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells.
Assuntos
Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Tubulina (Proteína)/química , Tubulina (Proteína)/fisiologia , Animais , Química Encefálica , Simulação por Computador , Dimerização , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fluorescência , Pulmão/citologia , Microscopia de Fluorescência/métodos , Modelos Estruturais , Fotografação/instrumentação , Fotografação/métodos , Salamandridae , Sensibilidade e Especificidade , Processos Estocásticos , Suínos , Tubulina (Proteína)/ultraestruturaRESUMO
The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphase, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle.