scholarly journals Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

2021 ◽  
Author(s):  
Ian Daniel Wolff ◽  
Jeremy Alden Hollis ◽  
Sarah Marie Wignall
Keyword(s):  
Adaptor Protein ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
C Elegans ◽  
Insight Into

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force. Instead, the kinesin-12 family motor KLP-18 performs this function. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments

1999 ◽  
Vol 112 (22) ◽  
pp. 3943-3954 ◽  
Author(s):  
V. Assmann ◽  
D. Jenkinson ◽  
J.F. Marshall ◽  
I.R. Hart
Keyword(s):  
Actin Filaments ◽  
Human Cancer ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
Projection Domain ◽  
Hyaluronan Receptor

We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca(2+)-dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574–602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.

scholarly journals Competitive Cofactor Recruitment by Orphan Receptor Hepatocyte Nuclear Factor 4α1: Modulation by the F Domain

2002 ◽  
Vol 22 (6) ◽  
pp. 1626-1638 ◽  
Author(s):  
Michael D. Ruse ◽  
Martin L. Privalsky ◽  
Frances M. Sladek
Keyword(s):  
Direct Interaction ◽  
Orphan Receptor ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Interaction Domain ◽  
The Status

ABSTRACT For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4α1 (HNF4α1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4α1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4α-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4α1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4α1 in vitro and repress HNF4α1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4α1 and GRIP1, CBP, or p300, SMRT can titer out HNF4α1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4α can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.

scholarly journals Insight into the interaction between the RNA helicase CGH-1 and EDC-3 and its implications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yong Zhang ◽  
Ke Wang ◽  
Kanglong Yang ◽  
Yunyu Shi ◽  
Jingjun Hong
Keyword(s):  
Homo Sapiens ◽  
Binding Mode ◽  
Translation Inhibition ◽  
C Elegans ◽  
Dead Box ◽  
Binding Interface ◽  
Body Components ◽  
Insight Into

AbstractPrevious studies indicated that the P-body components, CGH-1 and EDC-3 may play a crucial role in the regulation of lifespan in Caenorhabditis elegans. Homo sapiens DDX6 or Saccharomyces cerevisiae Dhh1p (CGH-1 in C. elegans) could form complexes with EDC3 (Edc3p in yeast), respectively, which is significant for translation inhibition and mRNA decay. However, it is currently unclear how CGH-1 can be recognized by EDC-3 in C. elegans. Here, we provided structural and biochemical insights into the interaction between CGH-1 and EDC-3. Combined with homology modeling, mutation, and ITC assays, we uncovered an interface between CGH-1 RecA2 domain and EDC-3 FDF-FEK. Additionally, GST-pulldown and co-localization experiments confirmed the interaction between CGH-1 and EDC-3 in vitro and in vivo. We also analyzed PATR-1-binding interface on CGH-1 RecA2 by ITC assays. Moreover, we unveiled the similarity and differences of the binding mode between EDC-3 and CAR-1 or PATR-1. Taken together, these findings provide insights into the recognition of DEAD-box protein CGH-1 by EDC-3 FDF-FEK motif, suggesting important functional implications.

scholarly journals Mechanical Stretch Inhibition Sensitizes Proprioceptors to Compressive Stresses

2021 ◽  
Author(s):  
Ravi Das ◽  
Li-Chun Lin ◽  
Frederic Català-Castro ◽  
Nawaphat Malaiwong ◽  
Neus Sanfeliu ◽  
...  
Keyword(s):  
Dimensional Space ◽  
Mechanical Stretch ◽  
C Elegans ◽  
Compressive Stresses ◽  
Tension And Compression ◽  
Low Dimensional ◽  
Spectrin Network ◽  
Insight Into

AbstractA repetitive gait cycle is an archetypical component within the behavioural repertoire of many if not all animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motorprogram during locomotion and thus leads to a periodic orbit in a low dimensional space. Here, we investigate the mechanics, molecules and neurons responsible for proprioception in Caenorhabditis (C.) elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive ‘stretch’ reception, we found that proprioceptors activated under compressive stresses in vivo and in vitro, and speculate that this property is conserved across function and species.

unc-53 controls longitudinal migration in C. elegans

Development ◽  
2002 ◽  
Vol 129 (14) ◽  
pp. 3367-3379 ◽  
Author(s):  
Eve Stringham ◽  
Nathalie Pujol ◽  
Joel Vandekerckhove ◽  
Thierry Bogaert
Keyword(s):  
Binding Sites ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Actin Binding ◽  
C Elegans ◽  
Guidance Cues ◽  
Preferred Direction ◽  
Excretory Canals ◽  
Novel Protein

Cell migration and outgrowth are thought to be based on analogous mechanisms that require repeated cycles of process extension, reading and integration of multiple directional signals, followed by stabilisation in a preferred direction, and renewed extension. We have characterised a C. elegans gene, unc-53, that appears to act cell autonomously in the migration and outgrowth of muscles, axons and excretory canals. Abrogation of unc-53 function disrupts anteroposterior outgrowth in those cells that normally express the gene. Conversely, overexpression of unc-53 in bodywall muscles leads to exaggerated outgrowth. UNC-53 is a novel protein conserved in vertebrates that contains putative SH3- and actin-binding sites. unc-53 interacts genetically with sem-5 and we demonstrated a direct interaction in vitro between UNC-53 and the SH2-SH3 adaptor protein SEM-5/GRB2. Thus, unc-53 is involved in longitudinal navigation and might act by linking extracellular guidance cues to the intracellular cytoskeleton.

scholarly journals The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

1999 ◽  
Vol 147 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Vicki Mountain ◽  
Calvin Simerly ◽  
Louisa Howard ◽  
Asako Ando ◽  
Gerald Schatten ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Cross Linking ◽  
Simultaneous Inhibition ◽  
Balance Of Forces ◽  
And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

scholarly journals Centromere function on minichromosomes isolated from budding yeast.

1993 ◽  
Vol 4 (8) ◽  
pp. 859-870 ◽  
Author(s):  
J Kingsbury ◽  
D Koshland
Keyword(s):  
Budding Yeast ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Microtubule Binding ◽  
Specific Factors ◽  
Stable Core ◽  
Insight Into ◽  
Binding Component

Centromeres are a complex of centromere DNA (CEN DNA) and specific factors that help mediate microtubule-dependent movement of chromosomes during mitosis. Minichromosomes can be isolated from budding yeast in a way that their centromeres retain the ability to bind microtubules in vitro. Here, we use the binding of these minichromosomes to microtubules to gain insight into the properties of centromeres assembled in vivo. Our results suggest that neither chromosomal DNA topology nor proximity of telomeres influence the cell's ability to assemble centromeres with microtubule-binding activity. The microtubule-binding activity of the minichromosome's centromere is stable in the presence of competitor CEN DNA, suggesting that the complex between the minichromosome CEN DNA and proteins directly bound to it is very stable. The efficiency of centromere binding to microtubules is dependent upon the concentration of microtubule polymer and is inhibited by ATP. These properties are similar to those exhibited by mechanochemical motors. The binding of minichromosomes to microtubules can be inactivated by the presence of 0.2 M NaCl and then reactivated by restoring NaCl to 0.1 M. In 0.2 M NaCl, some centromere factor(s) bind to microtubules, whereas other(s) apparently remain bound to the minichromosome's CEN DNA. Therefore, the yeast centromere appears to consist of two domains: the first consists of a stable core containing CEN DNA and CEN DNA-binding proteins; the second contains a microtubule-binding component(s). The molecular functions of this second domain are discussed.

scholarly journals Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly

2018 ◽  
Vol 29 (6) ◽  
pp. 722-735 ◽  
Author(s):  
Sarah Bianchi ◽  
Kacper B. Rogala ◽  
Nicola J. Dynes ◽  
Manuel Hilbert ◽  
Sebastian A. Leidel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Organelle Assembly ◽  
In Vivo Analysis ◽  
Cilia And Flagella

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans. SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5–microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

scholarly journals SH2B3, Transcribed by STAT1, Promotes Glioblastoma Progression Through Transducing IL-6/gp130 Signaling to Activate STAT3 Signaling

2021 ◽  
Vol 9 ◽  
Author(s):  
Shan Cai ◽  
Jian-xiang Lu ◽  
Yan-pei Wang ◽  
Chao-jia Shi ◽  
Tian Yuan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Adaptor Protein ◽  
Transcriptional Level ◽  
Self Renewal ◽  
Stat3 Signaling ◽  
Signaling Axis ◽  
Prognosis Marker ◽  
Insight Into

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. The aberrant activation of STAT3 commonly occurs in GBM and is a key player in GBM tumorigenesis. Yet, the aberrant activation of STAT3 signaling is not fully understood. Here, we report that SH2B adaptor protein 3 (SH2B3) is highly expressed in GBM and preferentially expressed in GBM stem cells (GSCs). Moreover, SH2B3 high expression predicts worse survival of GBM patients. Targeting SH2B3 considerably impairs GBM cell proliferation, migration, and GSCs’ self-renewal in vitro as well as xenograft tumors growth in vivo. Additionally, we provide evidence suggesting that STAT1 directly binds to the promoter of SH2B3 and activates SH2B3 expression in the transcriptional level. Functionally, SH2B3 facilitates GBM progression via physically interacting with gp130 and acting as an adaptor protein to transduce IL-6/gp130/STAT3 signaling. Together, our work firstly uncovers that the STAT1/SH2B3/gp130/STAT3 signaling axis plays critical roles in promoting GBM progression and provides insight into new prognosis marker and therapeutic target in GBM.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

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