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1.
Biophys J ; 121(10): 1813-1822, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35450824

RESUMO

Cytoskeletal filaments, such as microtubules and actin filaments, play important roles in the mechanical integrity of cells and the ability of cells to respond to their environment. Measuring the mechanical properties of cytoskeletal structures is crucial for gaining insight into intracellular mechanical stresses and their role in regulating cellular processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring filament deformations, such as Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature distributions can be used as an alternative tool to quantify biofilament deformations, and investigate how the apparent stiffness of filaments depends on the resolution and noise of the imaging system. We present analytical calculations of the scaling curvature distributions as a function of filament discretization, and test our predictions by comparing Monte Carlo simulations with results from existing techniques. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of nonfunctional motors, and calculate the persistence length of these filaments. The presented curvature analysis is significantly more accurate compared with existing approaches for small data sets, and can be readily applied to both in vitro and in vivo filament data through the use of the open-source codes we provide.


Assuntos
Citoesqueleto de Actina , Citoesqueleto , Citoesqueleto de Actina/química , Microscopia de Fluorescência , Microtúbulos , Estresse Mecânico
2.
J Cell Sci ; 133(4)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31964706

RESUMO

The actin cytoskeleton and active membrane trafficking machinery are essential for polarized cell growth. To understand the interactions between myosin XI, vesicles and actin filaments in vivo, we performed fluorescence recovery after photobleaching and showed that the dynamics of myosin XIa at the tip of the spreading earthmoss Physcomitrella patens caulonemal cells are actin-dependent and that 50% of myosin XI is bound to vesicles. To obtain single-particle information, we used variable-angle epifluorescence microscopy in protoplasts to demonstrate that protein myosin XIa and VAMP72-labeled vesicles localize in time and space over periods lasting only a few seconds. By tracking data with Hidden Markov modeling, we showed that myosin XIa and VAMP72-labeled vesicles exhibit short runs of actin-dependent directed transport. We also found that the interaction of myosin XI with vesicles is short-lived. Together, this vesicle-bound fraction, fast off-rate and short average distance traveled seem be crucial for the dynamic oscillations observed at the tip, and might be vital for regulation and recycling of the exocytosis machinery, while simultaneously promoting vesicle focusing and vesicle secretion at the tip, necessary for cell wall expansion.


Assuntos
Actinas , Bryopsida , Citoesqueleto de Actina , Actinas/genética , Bryopsida/genética , Exocitose , Miosinas/genética
3.
Plant Physiol ; 187(4): 2509-2529, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34890463

RESUMO

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell's tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin's local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.


Assuntos
Actinas/metabolismo , Bryopsida/fisiologia , Miosinas/metabolismo , Proteínas de Plantas/metabolismo , Organelas/fisiologia
4.
Proc Natl Acad Sci U S A ; 116(52): 26564-26570, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31822619

RESUMO

Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a koff that matched single-molecule measurements and measured a 2D kon for membrane-bound kinesin-1 motors binding to the microtubule. This kon is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this kon is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2D kon predicts that long-range transport of 100-nm-diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.

5.
Plant Mol Biol ; 107(4-5): 227-244, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33825083

RESUMO

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.


Assuntos
Briófitas/crescimento & desenvolvimento , Meristema/crescimento & desenvolvimento , Células Vegetais/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Tubo Polínico/crescimento & desenvolvimento , Citoesqueleto de Actina/metabolismo , Algoritmos , Briófitas/citologia , Briófitas/metabolismo , Meristema/citologia , Meristema/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Tubo Polínico/citologia , Tubo Polínico/metabolismo
6.
J Cell Sci ; 132(20)2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533971

RESUMO

The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.


Assuntos
Matriz Extracelular/metabolismo , Fase G1 , Podossomos/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Matriz Extracelular/genética , Técnicas de Inativação de Genes , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Podossomos/genética , Fase S
7.
Anal Biochem ; 596: 113626, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081618

RESUMO

We have developed new software, Re-track, that will quantify the rates of retraction and protrusion of structures emanating from the central core of a cell, such as neurites or filopodia. Re-Track, uses time-lapse images of cells in TIFF format and calculates the velocity of retraction or protrusion of a selected structure. The software uses a flexible moving boundary and has the ability to correct this boundary throughout analysis. Re-Track is fast, platform independent, and user friendly, and it can be used to follow biological events such as changes in neuronal connections, tip-growing cells such as moss, adaptive migration of cells, and similar behavior in non-biological systems.


Assuntos
Neuritos/química , Pseudópodes/química , Software , Animais , Diferenciação Celular , Células Cultivadas , Neuritos/metabolismo , Imagem Óptica , Células PC12 , Pseudópodes/metabolismo , Ratos
8.
Biophys J ; 116(6): 1115-1126, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30824116

RESUMO

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this "K-loop" is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.


Assuntos
Cinesinas/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Modelos Biológicos
9.
Plant Physiol ; 176(1): 352-363, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28972078

RESUMO

F-actin has been shown to be essential for tip growth in an array of plant models, including Physcomitrella patens One hypothesis is that diffusion can transport secretory vesicles, while actin plays a regulatory role during secretion. Alternatively, it is possible that actin-based transport is necessary to overcome vesicle transport limitations to sustain secretion. Therefore, a quantitative analysis of diffusion, secretion kinetics, and cell geometry is necessary to clarify the role of actin in polarized growth. Using fluorescence recovery after photobleaching analysis, we first show that secretory vesicles move toward and accumulate at the tip in an actin-dependent manner. We then depolymerized F-actin to decouple vesicle diffusion from actin-mediated transport and measured the diffusion coefficient and concentration of vesicles. Using these values, we constructed a theoretical diffusion-based model for growth, demonstrating that with fast-enough vesicle fusion kinetics, diffusion could support normal cell growth rates. We further refined our model to explore how experimentally extrapolated vesicle fusion kinetics and the size of the secretion zone limit diffusion-based growth. This model predicts that diffusion-mediated growth is dependent on the size of the region of exocytosis at the tip and that diffusion-based growth would be significantly slower than normal cell growth. To further explore the size of the secretion zone, we used a cell wall degradation enzyme cocktail and determined that the secretion zone is smaller than 6 µm in diameter at the tip. Taken together, our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth.


Assuntos
Actinas/metabolismo , Bryopsida/citologia , Polaridade Celular , Vesículas Secretórias/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bryopsida/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Difusão , Cinética , Modelos Biológicos , Polimerização/efeitos dos fármacos , Vesículas Secretórias/efeitos dos fármacos , Tiazolidinas/farmacologia
10.
Biophys J ; 114(5): 1153-1164, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29539401

RESUMO

Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a 3D computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorophore, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, Saccharomyces pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite or DCMS-can also perform fluorescence dynamics assays, such as number and brightness, fluorescence correlation spectroscopy, and raster image correlation spectroscopy, and could help shape the way these techniques are interpreted.


Assuntos
Bryopsida/citologia , Recuperação de Fluorescência Após Fotodegradação/métodos , Membrana Celular/metabolismo , Forma Celular , Fenômenos Ópticos
11.
Biophys J ; 110(12): 2568-2576, 2016 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-27332116

RESUMO

Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms.


Assuntos
Mecanotransdução Celular/fisiologia , Miosinas/metabolismo , Humanos , Modelos Moleculares
12.
Proc Natl Acad Sci U S A ; 109(51): 20814-9, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23213204

RESUMO

Intracellular transport of cargo particles is performed by multiple motors working in concert. However, the mechanism of motor association to cargos is unknown. It is also unknown how long individual motors stay attached, how many are active, and how multimotor cargos would navigate a densely crowded filament with many other motors. Prior theoretical and experimental biophysical model systems of intracellular cargo have assumed fixed teams of motors transporting along bare microtubules or microtubules with fixed obstacles. Here, we investigate a regime of cargos transporting along microtubules crowded with free motors. Furthermore, we use cargos that are able to associate or dissociate motors as it translocates. We perform in vitro motility reconstitution experiments with high-resolution particle tracking. Our model system consists of a quantum dot cargo attached to kinesin motors, and additional free kinesin motors that act as traffic along the microtubule. Although high densities of kinesin motors hinder forward motion, resulting in a lower velocity, the ability to associate motors appears to enhance the run length and attachment time of the quantum dot, improving overall cargo transport. These results suggest that cargos that can associate new motors as they transport could overcome traffic jams.


Assuntos
Axônios/metabolismo , Biofísica/métodos , Citoplasma/metabolismo , Dineínas/química , Cinesinas/química , Microtúbulos/metabolismo , Animais , Transporte Biológico , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde/metabolismo , Cinesinas/metabolismo , Cinética , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Polietilenoglicóis/química , Ligação Proteica , Pontos Quânticos , Suínos , Temperatura , Fatores de Tempo
13.
J Integr Plant Biol ; 57(1): 106-19, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25351786

RESUMO

In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.


Assuntos
Actinas/metabolismo , Bryopsida/metabolismo , Bryopsida/efeitos da radiação , Cloroplastos/metabolismo , Cinesinas/metabolismo , Luz , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/efeitos da radiação , Cloroplastos/efeitos da radiação , Técnicas de Silenciamento de Genes , Microtúbulos/metabolismo , Microtúbulos/efeitos da radiação , Movimento
14.
Biophys J ; 107(8): 1896-1904, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25418170

RESUMO

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.


Assuntos
Cinesinas/química , Microtúbulos/química , Animais , Drosophila , Cinesinas/classificação , Cinesinas/metabolismo , Cinética , Camundongos , Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Xenopus
15.
Plant J ; 73(3): 417-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23020796

RESUMO

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F-actin-associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F-actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F-actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F-actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F-actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI-associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization-dependent motility in the moss cytoplasm, where myosin XI-associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI-associated vesicular structures and F-actin polymerization-driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F-actin in tip growing cells.


Assuntos
Actinas/metabolismo , Bryopsida/crescimento & desenvolvimento , Miosinas/metabolismo , Bryopsida/citologia , Bryopsida/metabolismo
16.
Plant Cell ; 23(10): 3696-710, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22003077

RESUMO

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Bryopsida/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fatores de Despolimerização de Actina/genética , Sequência de Bases , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Bryopsida/ultraestrutura , Citosol/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , RNA de Plantas/genética , Genética Reversa , Análise de Sequência de DNA , Fatores de Tempo
17.
bioRxiv ; 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38464088

RESUMO

In this protocol, we describe steps to design, fabricate and use the Device for Axon and Cancer cell Interaction Testing (DACIT) in 2D and in 3D. In the first section, we detail steps to generate the mask, the master and the smooth-on mold. Next, we describe the step-by-step protocol for fabricating the DACIT, loading sensory neurons and cancer cells in 2D or 3D. We compare axonogenesis using PC-12 cell line and primary embryonic or adult sensory neurons, demonstrating the superior neurite growth in primary cells. We demonstrate DACIT can be used to compartmentalize neuronal soma and axons and expose them to different conditions, or to form a temporary gradient of neurotransmitter. Finally, we show that DACIT can be used to measure spheroid invasion in 3D in the presence of axons.

18.
Small ; 9(20): 3374-84, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23677651

RESUMO

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperm's journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.


Assuntos
Movimento Celular , Microfluídica/métodos , Espermatozoides/citologia , Animais , Separação Celular , Simulação por Computador , Humanos , Masculino , Camundongos , Fatores de Tempo
19.
Plant Cell ; 22(6): 1868-82, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20525854

RESUMO

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5' untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.


Assuntos
Regiões 5' não Traduzidas , Bryopsida/genética , Miosinas/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Actinas/metabolismo , Bryopsida/crescimento & desenvolvimento , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miosinas/genética , Proteínas de Plantas/genética , Brotos de Planta/genética , Interferência de RNA , RNA de Plantas/genética
20.
BMC Plant Biol ; 12: 70, 2012 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-22594499

RESUMO

BACKGROUND: In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. RESULTS: We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. CONCLUSIONS: To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles from different tip growing plant cells, thus enhancing our understanding of the mechanisms of plant polarized cell growth.


Assuntos
Bryopsida/citologia , Organelas/metabolismo , Células Vegetais/metabolismo , Bryopsida/química , Bryopsida/genética , Bryopsida/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Organelas/química , Organelas/genética , Células Vegetais/química
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