Robust patterns in the stochastic organization of filopodia

Husainy, Asma N; Morrow, Anne A.; Perkins, Theodore J.; Lee, Jonathan M

doi:10.1186/1471-2121-11-86

Cited by 20 publications

(24 citation statements)

References 32 publications

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“…We extracted the lengths of filopodia from total internal reflection fluorescence (TIRF) images and found they adopted a log-normal distribution (Fig. 1F), as previously reported for both filopodia (14) and neuronal spines (15). The mode length of this distribution is 5.66 ± 0.14 μm compared with 3.22 ± 0.04 μm in cells transfected with GFP-MYO6.…”

Section: Significancementioning

confidence: 67%

Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Masters

Buß

2017

Proc. Natl. Acad. Sci. U.S.A.

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Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P 2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.is a monomeric motor protein with a large working stroke (1), which takes processive 36-nm steps along actin filaments in vitro when dimerized (2). Uniquely among myosin motors, it takes these steps toward the minus end of actin filaments (3). In cells and tissues, MYO6 has been implicated in an ever-growing list of functions, including secretion, endocytosis, cell migration, stereocilia maintenance, and autophagy (4). It mediates these functions through interactions with a wide array of cargo adaptor proteins that bind to the tail region of MYO6, thereby localizing and potentially activating the motor through conformational change and unfolding of the lever arm (5) mediated by calcium (6). However, how the mechanical properties (for instance step size, duty ratio, stall force, etc.) and in particular the directionality of MYO6 relate to its biological roles is poorly understood.Isolated, purified MYO6 is primarily monomeric (1), but can dimerize in vitro through interactions between the cargo binding domain and adaptor molecules (2, 7). Whether any of these adaptors are capable of inducing functional MYO6 dimers in cells is not known and there has been little definitive evidence as to whether MYO6 functions as a monomer or as a processive dimer in cells and how motor processivity relates to its biological function (8,...

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Section: Significancementioning

confidence: 67%

Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Masters

Buß

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This approach is tedious and slow, limiting the potential size of studies and their statistical power. Our previous work shows that there is high variability in filopodia characteristics, even among genetically identical cells in identical culture conditions [5]. Thus to study filopodia under several different conditions can require tens, if not hundreds, of manually annotated images.…”

Section: Introductionmentioning

confidence: 99%

“…Our recent work suggests that filopodia sizes in non-neural cells are lognormally distributed [5]. The few, longest filopodia are not representative of the majority of the filopodia distribution, and thus we must detect all or nearly all filopodia to accurately assess the length distribution.…”

Section: Introductionmentioning

confidence: 99%

“…We validated FiloDetect on the non-transformed rodent cell line Rat2 and mouse melanoma cell line B16F1. The Rat2 images used to test the algorithm have been previously manually annotated for filopodia length and number [5], allowing us to assess the accuracy. The B16F1 images were annotated newly for this study.…”

Section: Introductionmentioning

confidence: 99%

“…We were interested in this question because of several lines of evidence implicating a role for PI4KIII β in breast cancer and filopodia production: it is activated by eEF1A2 (eukaryotic elongation factor 1 alpha 2) [14], which is amplified in approximately two-thirds of breast tumours [15,16]; it was recently identified as a putative breast cancer driver gene, in a large-scale copy number and gene expression analysis of 2000 breast tumours [17]; and ectopic expression of PI4KIII β in fibroblast cells increases filopodia number and length [5,14]. Thus, we hypothesized that PI4KIII β may drive filopodia formation in breast cancer cells, potentially enhancing their invasivenes.…”

Section: Introductionmentioning

confidence: 99%

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FiloDetect: automatic detection of filopodia from fluorescence microscopy images

et al. 2013

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BackgroundFilopodia are small cellular projections that help cells to move through and sense their environment. Filopodia play crucial roles in processes such as development and wound-healing. Also, increases in filopodia number or size are characteristic of many invasive cancers and are correlated with increased rates of metastasis in mouse experiments. Thus, one possible route to developing anti-metastatic therapies is to target factors that influence the filopodia system. Filopodia can be detected by eye using confocal fluorescence microscopy, and they can be manually annotated in images to quantify filopodia parameters. Although this approach is accurate, it is slow, tedious and not entirely objective. Manual detection is a significant barrier to the discovery and quantification of new factors that influence the filopodia system.ResultsHere, we present FiloDetect, an automated tool for detecting, counting and measuring the length of filopodia in fluorescence microscopy images. The method first segments the cell from the background, using a modified triangle threshold method, and then extracts the filopodia using a series of morphological operations. We verified the accuracy of FiloDetect on Rat2 and B16F1 cell images from three different labs, showing that per-cell filopodia counts and length estimates are highly correlated with the manual annotations. We then used FiloDetect to assess the role of a lipid kinase on filopodia production in breast cancer cells. Experimental results show that PI4KIII β expression leads to an increase in filopodia number and length, suggesting that PI4KIII β is involved in driving filopodia production.ConclusionFiloDetect provides accurate and objective quantification of filopodia in microscopy images, and will enable large scale comparative studies to assess the effects of different genetic and chemical perturbations on filopodia production in different cell types, including cancer cell lines.

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Do Cell Membranes Flow Like Honey or Jiggle Like Jello?

Cohen

Shi

2019

BioEssays

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Cell membranes experience frequent stretching and poking: from cytoskeletal elements, from osmotic imbalances, from fusion and budding of vesicles, and from forces from the outside. Are the ensuing changes in membrane tension localized near the site of perturbation, or do these changes propagate rapidly through the membrane to distant parts of the cell, perhaps as a mechanical mechanism of long‐range signaling? Literature statements on the timescale for membrane tension to equilibrate across a cell vary by a factor of ≈106. This study reviews and discusses how apparently contradictory findings on tension propagation in cells can be evaluated in the context of 2D hydrodynamics and poroelasticity. Localization of tension in the cell membrane is likely critical in governing how membrane forces gate ion channels, set the subcellular distribution of vesicle fusion, and regulate the dynamics of cytoskeletal growth. Furthermore, in this study, it is proposed that cells can actively regulate the degree to which membrane tension propagates by modulating the density and arrangement of immobile transmembrane proteins. Also see the video abstract here https://youtu.be/T6K7AIAqqBs.

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Robust patterns in the stochastic organization of filopodia

Cited by 20 publications

References 32 publications

Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

FiloDetect: automatic detection of filopodia from fluorescence microscopy images

Do Cell Membranes Flow Like Honey or Jiggle Like Jello?

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