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1.
iScience ; 27(4): 109440, 2024 Apr 19.
Article in English | MEDLINE | ID: mdl-38510137

ABSTRACT

Plasma membrane-associated platforms (PMAPs) form at specific sites of plasma membrane by scaffolds including ERC1 and Liprin-α1. We identify a mechanism regulating PMAPs assembly, with consequences on motility/invasion. Silencing Ser/Thr kinase DYRK3 in invasive breast cancer cells inhibits their motility and invasive capacity. Similar effects on motility were observed by increasing DYRK3 levels, while kinase-dead DYRK3 had limited effects. DYRK3 overexpression inhibits PMAPs formation and has negative effects on stability of lamellipodia and adhesions in migrating cells. Liprin-α1 depletion results in unstable lamellipodia and impaired cell motility. DYRK3 causes increased Liprin-α1 phosphorylation. Increasing levels of Liprin-α1 rescue the inhibitory effects of DYRK3 on cell spreading, suggesting that an equilibrium between Liprin-α1 and DYRK3 levels is required for lamellipodia stability and tumor cell motility. Our results show that DYRK3 is relevant to tumor cell motility, and identify a PMAP target of the kinase, highlighting a new mechanism regulating cell edge dynamics.

2.
Dev Cell ; 58(19): 1880-1897.e11, 2023 Oct 09.
Article in English | MEDLINE | ID: mdl-37643612

ABSTRACT

The dual-specificity kinase DYRK3 controls the formation and dissolution of multiple biomolecular condensates, regulating processes including stress recovery and mitotic progression. Here, we report that DYRK3 functionally interacts with proteins associated with endoplasmic reticulum (ER) exit sites (ERESs) and that inhibition of DYRK3 perturbs the organization of the ERES-Golgi interface and secretory trafficking. DYRK3-mediated regulation of ERES depends on the N-terminal intrinsically disordered region (IDR) of the peripheral membrane protein SEC16A, which co-phase separates with ERES components to form liquid-like condensates on the surface of the ER. By modulating the liquid-like properties of ERES, we show that their physical state is essential for functional cargo trafficking through the early secretory pathway. Our findings support a mechanism whereby phosphorylation by DYRK3 and its reversal by serine-threonine phosphatases regulate the material properties of ERES to create a favorable physicochemical environment for directional membrane traffic in eukaryotic cells.

3.
Methods Mol Biol ; 2623: 187-200, 2023.
Article in English | MEDLINE | ID: mdl-36602687

ABSTRACT

Optical trapping of organelles inside cells is a powerful technique for directly measuring the forces generated by motor proteins when they are transporting the organelle in the form of a "cargo". Such experiments provide an understanding of how multiple motors (similar or dissimilar) function in their endogenous environment. Here we describe the use of latex bead phagosomes ingested by macrophage cells as a model cargo for optical trap-based force measurements. A protocol for quantitative force measurements of microtubule-based motors (dynein and kinesins) inside macrophage cells is provided.


Subject(s)
Kinesins , Phagosomes , Microspheres , Kinesins/metabolism , Phagosomes/metabolism , Dyneins/metabolism , Biological Transport , Microtubules/metabolism
4.
Cell Syst ; 13(6): 454-470.e15, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35613616

ABSTRACT

RNA concentration homeostasis involves coordinating RNA abundance and synthesis rates with cell size. Here, we study this in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations generally remain highly constant. Perturbations that would be expected to increase nuclear mRNA levels, including those targeting nuclear mRNA degradation or export, result in downregulation of RNA synthesis. This is associated with reduced abundance of transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (RNA Pol II) itself. Acute perturbations, elevation of nuclear mRNA levels, and mathematical modeling indicate that mammalian cells achieve robust mRNA concentration homeostasis by the mRNA-based negative feedback on transcriptional activity in the nucleus. This ultimately acts to coordinate RNA Pol II abundance with nuclear mRNA degradation and export rates and may underpin the scaling of mRNA abundance with cell size.


Subject(s)
RNA Polymerase II , RNA, Nuclear , Animals , Feedback , Homeostasis/genetics , Humans , Mammals/genetics , RNA , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/genetics
5.
Science ; 367(6483): 1193-1194, 2020 03 13.
Article in English | MEDLINE | ID: mdl-32165570
6.
Nature ; 559(7713): 211-216, 2018 07.
Article in English | MEDLINE | ID: mdl-29973724

ABSTRACT

Liquid-liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis.


Subject(s)
Mitosis , Organelles/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Anaphase-Promoting Complex-Cyclosome/metabolism , Cytoplasm/metabolism , Cytoplasmic Granules/metabolism , HEK293 Cells , HeLa Cells , Humans , Nuclear Envelope/metabolism , Poly(A)-Binding Protein I/metabolism , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/biosynthesis , Protein Transport , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/biosynthesis , Solubility , Spindle Apparatus/metabolism , Stress, Physiological
7.
Curr Biol ; 28(9): 1460-1466.e4, 2018 05 07.
Article in English | MEDLINE | ID: mdl-29706510

ABSTRACT

How the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5-7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells.


Subject(s)
Dyneins/physiology , Kinesins/physiology , Phagosomes/physiology , Biological Transport , Cell Line , Dictyostelium , Dyneins/metabolism , Kinesins/metabolism , Microtubules/physiology , Models, Biological , Phagocytosis , Phagosomes/metabolism , Probability , Protein Transport/physiology
8.
Trends Cell Biol ; 23(11): 575-82, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23877011

ABSTRACT

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.


Subject(s)
Microtubules/metabolism , Molecular Motor Proteins/metabolism , Organelles/metabolism
9.
Cell ; 152(1-2): 172-82, 2013 Jan 17.
Article in English | MEDLINE | ID: mdl-23332753

ABSTRACT

Many cellular processes require large forces that are generated collectively by multiple cytoskeletal motor proteins. Understanding how motors generate force as a team is therefore fundamentally important but is poorly understood. Here, we demonstrate optical trapping at single-molecule resolution inside cells to quantify force generation by motor teams driving single phagosomes. In remarkable paradox, strong kinesins fail to work collectively, whereas weak and detachment-prone dyneins team up to generate large forces that tune linearly in strength and persistence with dynein number. Based on experimental evidence, we propose that leading dyneins in a load-carrying team take short steps, whereas trailing dyneins take larger steps. Dyneins in such a team bunch close together and therefore share load better to overcome low/intermediate loads. Up against higher load, dyneins "catch bond" tenaciously to the microtubule, but kinesins detach rapidly. Dynein therefore appears uniquely adapted to work in large teams, which may explain how this motor executes bewilderingly diverse cellular processes.


Subject(s)
Biological Transport , Dyneins/metabolism , Phagosomes/metabolism , Animals , Biomechanical Phenomena , Brain Chemistry , Cell Line , Dictyostelium , Dyneins/chemistry , Goats , Kinesins , Macrophages/metabolism , Mice , Microspheres , Microtubules/metabolism , Optical Tweezers
10.
Proc Natl Acad Sci U S A ; 106(46): 19381-6, 2009 Nov 17.
Article in English | MEDLINE | ID: mdl-19864630

ABSTRACT

Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.


Subject(s)
Dictyostelium/metabolism , Dyneins/metabolism , Endosomes/metabolism , Kinesins/metabolism , Microtubules/metabolism , Microspheres , Monte Carlo Method
11.
Biotechniques ; 46(7): 543-9, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19594454

ABSTRACT

Transport of intracellular organelles along the microtubule cytoskeleton occurs in a bidirectional manner due to opposing activity of microtubule-associated motor proteins of the kinesin and dynein families. Regulation of this opposing activity and the resultant motion is believed to generate a polarized distribution of many organelles within the cell. The bidirectional motion can be reconstituted on in vitro assembled microtubules using organelles extracted from cells. This provides an opportunity to understand the regulation of intracellular transport through quantitative analysis of the motion of organelles in a controlled environment. Such analysis requires the use of polarity-labeled microtubules to resolve the plus and minus components of bidirectional motion. However, existing methods of in vitro microtubule polarity labeling are unsuitable for high-resolution recording of motion. Here we present a simple and reliable method that uses avidin-coated magnetic beads to prepare microtubules labeled at the minus end. The microtubule polarity can be identified without any need for fluorescence excitation. We demonstrate video-rate high-resolution imaging of single cellular organelles moving along plus and minus directions on labeled microtubules. Quantitative analysis of this motion indicates that these organelles are likely to be driven by multiple dynein motors in vivo.


Subject(s)
Biological Assay/methods , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Staining and Labeling/methods , Animals , Cell Polarity , Cytoplasmic Vesicles/metabolism , Dictyostelium/cytology , Dictyostelium/metabolism , Dyneins/metabolism , Fluorescence , Goats , Kinesins/metabolism , Magnetics , Microspheres
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