scholarly journals Differential Effects of LIS1 On Processive Dynein-Dynactin Motility

2017 ◽  
Author(s):  
Pedro A. Gutierrez ◽  
Richard J. McKenney
Keyword(s):  
Single Molecule ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Dependent Manner ◽  
Regulatory Factor ◽  
Single Molecule Microscopy ◽  
Concentration Dependent Manner ◽  
Dynein Motor ◽  
Microtubule Motor ◽  
Microtubule Motor Protein

AbstractCytoplasmic dynein is the primary minus-end directed microtubule motor protein in cells. LIS1 is a highly conserved dynein regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor, and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein regulatory factor, acts to release dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we find that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bind up to two LIS1 dimers, and mutational analysis suggests LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhances DDB velocity in a concentration dependent manner, in contrast to observations of LIS1’s effects on the motility of isolated dynein. Thus, LIS1 exerts concentration dependent effects on dynein motility, and can synergize with dynactin to enhance processive movement in the absence of load.

The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2409-2419 ◽  
Author(s):  
M. McGrail ◽  
T.S. Hays
Keyword(s):  
Asymmetric Cell Division ◽  
Fruit Fly ◽  
Cytoplasmic Dynein ◽  
Spindle Orientation ◽  
Chain Gene ◽  
Animal Development ◽  
Dynein Motor ◽  
Microtubule Motor ◽  
Two Stages ◽  
Oocyte Differentiation

During animal development cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In the fruit fly, Drosophila melanogaster, the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast which undergoes 4 synchronous divisions with incomplete cytokinesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, we show that cytoplasmic dynein is required at two stages of oogenesis. Early in oogenesis, dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The localization of dynein in mitotic cysts suggests spindle orientation is mediated by the microtubule motor cytoplasmic dynein. Later in oogenesis, dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the cytoplasmic dynein motor in cellular determination and differentiation.

scholarly journals The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1211-1220 ◽  
Author(s):  
Kristin L M Boylan ◽  
Thomas S Hays
Keyword(s):  
Intracellular Transport ◽  
De Novo ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Wing Development ◽  
Dynein Motor ◽  
Dynein Intermediate Chain ◽  
Microtubule Motor ◽  
Intermediate Chain

Abstract The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.

scholarly journals Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development

2020 ◽  
Author(s):  
Gokul Kesavan ◽  
Florian Raible ◽  
Mansi Gupta ◽  
Anja Machate ◽  
Dilara Yilmaz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gene Expression Pattern ◽  
Correlation Spectroscopy ◽  
Dependent Manner ◽  
Embryonic Patterning ◽  
Loss Of Function ◽  
Concentration Dependent Manner ◽  
Developmental Defects ◽  
Extracellular Signals

AbstractExtracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

scholarly journals The Third P-loop Domain in Cytoplasmic Dynein Heavy Chain Is Essential for Dynein Motor Function and ATP-sensitive Microtubule Binding

2003 ◽  
Vol 14 (4) ◽  
pp. 1355-1365 ◽  
Author(s):  
Andre Silvanovich ◽  
Min-gang Li ◽  
Madeline Serr ◽  
Sarah Mische ◽  
Thomas S. Hays
Keyword(s):  
Heavy Chain ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Sequence Comparisons ◽  
Microtubule Binding ◽  
Dynein Heavy Chain ◽  
Dynein Motor ◽  
Microtubule Binding Domain ◽  
Atp Binding And Hydrolysis ◽  
Loop Domains

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of theDrosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

scholarly journals Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

1996 ◽  
Vol 133 (3) ◽  
pp. 585-593 ◽  
Author(s):  
J Niclas ◽  
V J Allan ◽  
R D Vale
Keyword(s):  
Cell Cycle ◽  
Membrane Surface ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
A Cell ◽  
Cycle Dependent ◽  
Intermediate Chain

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

scholarly journals Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.

1994 ◽  
Vol 126 (6) ◽  
pp. 1475-1494 ◽  
Author(s):  
M Li ◽  
M McGrail ◽  
M Serr ◽  
T S Hays
Keyword(s):  
Heavy Chain ◽  
Maternal Effect ◽  
Nurse Cell ◽  
Cytoplasmic Dynein ◽  
Cdna Clones ◽  
Dynein Heavy Chain ◽  
Dynein Motor ◽  
Maternal Effect Gene ◽  
Microtubule Motor ◽  
Egg Chambers

The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte.

scholarly journals Nanometer-Resolution Long-term Tracking of Single Cargos Reveals Dynein Motor Mechanisms

2022 ◽  
Author(s):  
Chunte Sam Peng ◽  
Yunxiang Zhang ◽  
Qian Liu ◽  
G. Edward Marti ◽  
Yu-Wen Alvin Huang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Molecular Model ◽  
Cytoplasmic Dynein ◽  
Single Particle Tracking ◽  
Thermally Activated ◽  
Dynein Motor ◽  
Biochemical Measurements ◽  
Hydrolysis Of

Cytoplasmic dynein is essential for intracellular transport, but because of its complexity, we still do not fully understand how this 1.5 megadalton protein works. Here, we used novel optical probes that enable single-particle tracking (SPT) of individual cargos transported by dynein motors in live neurons over 900 μm. Analyses using the Fluctuation Theorem (FT) showed that the number of dynein molecules switches between 1-5 motors during the transport. Clearly resolved single-molecular steps revealed that the dwell times between individual steps were accurately described by an enzymatic cycle dominated by two equal and thermally-activated rate constants. Based on these data, we propose a new molecular model whereby each step requires the hydrolysis of 2 ATPs. The model is consistent with extensive structural, single-molecule and biochemical measurements.

scholarly journals Designing Single-Molecule Magnets as Drugs with Dual Anti-Inflammatory and Anti-Diabetic Effects

2020 ◽  
Vol 21 (9) ◽  
pp. 3146
Author(s):  
Arturo Navas ◽  
Fatin Jannus ◽  
Belén Fernández ◽  
Javier Cepeda ◽  
Marta Medina O’Donnell ◽  
...  
Keyword(s):  
Metal Complexes ◽  
Single Molecule ◽  
Raw 264.7 Cells ◽  
Cobalt Metal ◽  
No Production ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Anti Inflammatory

We have designed and synthesized two novel cobalt coordination compounds using bumetanide (bum) and indomethacin (ind) therapeutic agents. The anti-inflammatory effects of cobalt metal complexes with ind and bum were assayed in lipopolysaccharide stimulated RAW 264.7 macrophages by inhibition of nitric oxide production. Firstly, we determined the cytotoxicity and the anti-inflammatory potential of the cobalt compounds and ind and bum ligands in RAW 264.7 cells. Indomethacin-based metal complex was able to inhibit the NO production up to 35% in a concentration-dependent manner without showing cytotoxicity, showing around 6–37 times more effective than indomethacin. Cell cycle analysis showed that the inhibition of NO production was accompanied by a reversion of the differentiation processes in LPS-stimulated RAW 264.7 cells, due to a decreased of cell percentage in G0/G1 phase, with the corresponding increase in the number of cells in S phase. These two materials have mononuclear structures and show slow relaxation of magnetization. Moreover, both compounds show anti-diabetic activity with low in vitro cell toxicities. The formation of metal complexes with bioactive ligands is a new and promising strategy to find new compounds with high and enhanced biochemical properties and promises to be a field of great interest.

scholarly journals New insights into the mechanism of dynein motor regulation by lissencephaly-1

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Steven M Markus ◽  
Matthew G Marzo ◽  
Richard J McKenney
Keyword(s):  
Cognitive Impairment ◽  
Brain Development ◽  
Cytoplasmic Dynein ◽  
Brain Disease ◽  
Causative Gene ◽  
New Model ◽  
Dynein Motor ◽  
Microtubule Motor ◽  
Neuronal Transport

Lissencephaly (‘smooth brain’) is a severe brain disease associated with numerous symptoms, including cognitive impairment, and shortened lifespan. The main causative gene of this disease – lissencephaly-1 (LIS1) – has been a focus of intense scrutiny since its first identification almost 30 years ago. LIS1 is a critical regulator of the microtubule motor cytoplasmic dynein, which transports numerous cargoes throughout the cell, and is a key effector of nuclear and neuronal transport during brain development. Here, we review the role of LIS1 in cellular dynein function and discuss recent key findings that have revealed a new mechanism by which this molecule influences dynein-mediated transport. In addition to reconciling prior observations with this new model for LIS1 function, we also discuss phylogenetic data that suggest that LIS1 may have coevolved with an autoinhibitory mode of cytoplasmic dynein regulation.

scholarly journals Pac1/LIS1 promotes an uninhibited conformation of dynein that coordinates its localization and activity

2019 ◽  
Author(s):  
Matthew G. Marzo ◽  
Jacqueline M. Griswold ◽  
Steven M. Markus
Keyword(s):  
Single Molecule ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Extrinsic Factors ◽  
Temporal Precision ◽  
Negative Stain ◽  
Microtubule Motor ◽  
And Function ◽  
High Degree ◽  
In Cells

ABSTRACTCytoplasmic dynein is a minus end-directed microtubule motor that transports myriad cargos in various cell types and contexts. How dynein is regulated to perform all these activities with a high degree of spatial and temporal precision is unclear. Recent studies have revealed that human dynein-1 and dynein-2 can be regulated by a mechanism of autoinhibition, whereby intermolecular contacts limit motor activity. Whether this autoinhibitory mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its precise role in regulating cellular dynein activity remain unknown. Here, we use a combination of negative stain EM, single molecule motility assays, genetic, and cell biological techniques to show that the autoinhibitory conformation is conserved in budding yeast, and it plays an important role in coordinating dynein localization and function in cells. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast) plays an important role in regulating this autoinhibitory conformation of dynein. Specifically, our studies demonstrate that rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate localization and activity of dynein in cells.

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