scholarly journals Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling

2011 ◽  
Author(s):  
Einat Sadot ◽  
Christopher Staiger ◽  
Mohamad Abu-Abied
Keyword(s):  
Actin Filaments ◽  
Cytoplasmic Streaming ◽  
Dominant Negative ◽  
Expression Vectors ◽  
In Vitro Assays ◽  
Tail Domain ◽  
Myosin V ◽  
Organelle Movement ◽  
Original Proposal ◽  
Myosin Xi

In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells.   Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.

scholarly journals Autoregulation and Dual Stepping Mode of MYA2, an Arabidopsis Myosin XI Responsible for Cytoplasmic Streaming

2021 ◽  
Author(s):  
Takashi Haraguchi ◽  
Kohji Ito ◽  
Takamitsu Morikawa ◽  
Nao Shoji ◽  
Mitsuhiro Iwaki ◽  
...  
Keyword(s):  
Cytoplasmic Streaming ◽  
Force Measurement ◽  
Plant Cells ◽  
Transfer System ◽  
Tail Domain ◽  
Animal Cells ◽  
Myosin V ◽  
Myosin Xi ◽  
Stall Force

Abstract Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD), When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining TIRF microscopy and the FIONA method, revealed that the MYA2 processively moved on actin with three different step sizes: −28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes, hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2 − 3 pN). These results indicated that MYA2 is more flexible than the myosin V responsible for vesicle transport in animal cells. Such flexibility may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

scholarly journals Transport of ER vesicles on actin filaments in neurons by myosin V

1998 ◽  
Vol 111 (21) ◽  
pp. 3221-3234 ◽  
Author(s):  
J.S. Tabb ◽  
B.J. Molyneaux ◽  
D.L. Cohen ◽  
S.A. Kuznetsov ◽  
G.M. Langford
Keyword(s):  
Axonal Transport ◽  
Actin Filaments ◽  
Giant Axon ◽  
Vesicle Transport ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Tail Domain ◽  
Myosin V ◽  
Fast Axonal Transport

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for the dual filament model of vesicle transport.

scholarly journals Discovery of the fastest myosin, its amino acid sequence, and structural features

2021 ◽  
Author(s):  
Takeshi Haraguchi ◽  
Masanori Tamanaha ◽  
Kano Suzuki ◽  
Kohei Yoshimura ◽  
Takuma Imi ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cytoplasmic Streaming ◽  
Structural Features ◽  
Chara Corallina ◽  
Actin Binding ◽  
X Ray Crystallography ◽  
Binding Surface ◽  
Fast Movement ◽  
Myosin Xi ◽  
Loop 2

Cytoplasmic streaming with extremely high velocity (~70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 μm s−1, suggesting that an unknown myosin XI with a velocity of 70 μm −1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis.

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo

1994 ◽  
Vol 14 (12) ◽  
pp. 8191-8201
Author(s):  
A Dey ◽  
S Minucci ◽  
K Ozato
Keyword(s):  
Retinoic Acid ◽  
Dimethyl Sulfate ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Cell Extracts ◽  
Ec Cells ◽  
Beta 2 ◽  
Responsive Element ◽  
Receptor Beta

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

scholarly journals The Cooh-Terminal Domain of Myo2p, a Yeast Myosin V, Has a Direct Role in Secretory Vesicle Targeting

1999 ◽  
Vol 147 (4) ◽  
pp. 791-808 ◽  
Author(s):  
Daniel Schott ◽  
Jackson Ho ◽  
David Pruyne ◽  
Anthony Bretscher
Keyword(s):  
Secretory Vesicle ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Tail Domain ◽  
Myosin V ◽  
Direct Role ◽  
Rab Protein ◽  
Polarized Delivery ◽  
Actin Cables ◽  
Type V

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

scholarly journals Arabidopsis Myosin XIK Interacts with the Exocyst Complex to Facilitate Vesicle Tethering during Exocytosis

2020 ◽  
Author(s):  
Weiwei Zhang ◽  
Lei Huang ◽  
Chunhua Zhang ◽  
Christopher J. Staiger
Keyword(s):  
Mammalian Cells ◽  
Secretory Vesicle ◽  
Tail Domain ◽  
Spatiotemporal Resolution ◽  
Dynamic Regulation ◽  
Vesicle Tethering ◽  
Exocyst Complex ◽  
Myosin Xi ◽  
Myosin Motors

ABSTRACTMyosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely-growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to the SEC5B subunit of exocyst in vitro and functional fluorescently-tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the frequency and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes (CSCs) with high spatiotemporal resolution imaging and pair-wise colocalization analysis of myosin XIK, exocyst subunits and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the recruitment of exocyst to the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides new insights about the dynamic regulation of exocytosis in plants.

scholarly journals p110 CUX1 Cooperates with E2F Transcription Factors in the Transcriptional Activation of Cell Cycle-Regulated Genes

2008 ◽  
Vol 28 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Mary Truscott ◽  
Ryoko Harada ◽  
Charles Vadnais ◽  
François Robert ◽  
Alain Nepveu
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Affinity Purification ◽  
Gene Promoter ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Dominant Negative Mutant ◽  
Dna Polymerase Α ◽  
Reporter Assays

ABSTRACT The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase α gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase α gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

scholarly journals Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination

2018 ◽  
Vol 217 (10) ◽  
pp. 3497-3511 ◽  
Author(s):  
Wen Lu ◽  
Margot Lakonishok ◽  
Anna S. Serpinskaya ◽  
David Kirchenbüechler ◽  
Shuo-Chien Ling ◽  
...  
Keyword(s):  
Germ Cells ◽  
Cytoplasmic Streaming ◽  
Rna Binding ◽  
Early Stage ◽  
Rna Binding Protein ◽  
Myosin V ◽  
Actin Cortex ◽  
Microtubule Motor ◽  
Actin Motor

The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte—transport along microtubules and cytoplasmic streaming—it is unclear how individual kinesin-1 activities contribute to the posterior determination. We examined Staufen, an RNA-binding protein that is colocalized with osk mRNA, as a proxy of posterior determination, and we used mutants that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. We demonstrated that late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. We propose a model whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V–based cortical retention cooperate in posterior determination.

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