scholarly journals Gravin-associated kinase signaling networks coordinate γ-tubulin organization at mitotic spindle poles

2020 ◽  
Vol 295 (40) ◽  
pp. 13784-13797
Author(s):  
Paula J. Bucko ◽  
Irvin Garcia ◽  
Ridhima Manocha ◽  
Akansha Bhat ◽  
Linda Wordeman ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Subcellular Location ◽  
Asymmetric Distribution ◽  
Binding Partner ◽  
Mitotic Kinases ◽  
Spindle Poles ◽  
Biochemical Assays ◽  
Anchoring Protein ◽  
Intracellular Compartments ◽  
Genetic Deletion

Mitogenic signals that regulate cell division often proceed through multienzyme assemblies within defined intracellular compartments. The anchoring protein Gravin restricts the action of mitotic kinases and cell-cycle effectors to defined mitotic structures. In this report we discover that genetic deletion of Gravin disrupts proper accumulation and asymmetric distribution of γ-tubulin during mitosis. We utilize a new precision pharmacology tool, Local Kinase Inhibition, to inhibit the Gravin binding partner polo-like kinase 1 at spindle poles. Using a combination of gene-editing approaches, quantitative imaging, and biochemical assays, we provide evidence that disruption of local polo-like kinase 1 signaling underlies the γ-tubulin distribution defects observed with Gravin loss. Our study uncovers a new role for Gravin in coordinating γ-tubulin recruitment during mitosis and illuminates the mechanism by which signaling enzymes regulate this process at a distinct subcellular location.

scholarly journals Gravin-associated kinase signaling networks coordinate γ-tubulin organization at mitotic spindle poles

2020 ◽  
Author(s):  
Paula J. Bucko ◽  
Irvin Garcia ◽  
Ridhima Manocha ◽  
Akansha Bhat ◽  
Linda Wordeman ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Subcellular Location ◽  
Asymmetric Distribution ◽  
Binding Partner ◽  
Mitotic Kinases ◽  
Spindle Poles ◽  
Biochemical Assays ◽  
Anchoring Protein ◽  
Intracellular Compartments ◽  
Genetic Deletion

AbstractMitogenic signals that regulate cell division often proceed through multi-enzyme assemblies within defined intracellular compartments. The anchoring protein Gravin restricts the action of mitotic kinases and cell-cycle effectors to defined mitotic structures. In this report we discover that genetic deletion of Gravin disrupts proper accumulation and asymmetric distribution of γ-tubulin during mitosis. We utilize a new precision pharmacology tool, Local Kinase Inhibition (LoKI), to inhibit the Gravin binding partner polo-like kinase 1 (Plk1) at spindle poles. Using a combination of gene-editing approaches, quantitative imaging, and biochemical assays we provide evidence that disruption of local Plk1 signaling underlies the γ-tubulin distribution defects observed with Gravin loss. Our study uncovers a new role for Gravin in coordinating γ-tubulin recruitment during mitosis and illuminates the mechanism by which signaling enzymes regulate this process at a distinct subcellular location.

scholarly journals Quantitative imaging of intracellular nanoparticle exposure enables prediction of nanotherapeutic efficacy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qingqing Yin ◽  
Anni Pan ◽  
Binlong Chen ◽  
Zenghui Wang ◽  
Mingmei Tang ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Tumour Mass ◽  
Nanoparticle Exposure ◽  
Imaging Tool ◽  
Intracellular Compartments ◽  
Signal Output ◽  
Extracellular Microenvironment ◽  
Intracellular Targets ◽  
Ph Variations ◽  
Therapeutic Responses

AbstractNanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7–28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.

scholarly journals Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein

2020 ◽  
Vol 117 (32) ◽  
pp. 19254-19265
Author(s):  
Jianming Liu ◽  
Zhan-Peng Huang ◽  
Mao Nie ◽  
Gang Wang ◽  
William J. Silva ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Function ◽  
Neuromuscular Junctions ◽  
Linc Complex ◽  
Muscle Dystrophy ◽  
Microtubule Organizing Center ◽  
Anchoring Protein ◽  
Organizing Center ◽  
Genetic Deletion

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy inmdxmice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

scholarly journals Curcumin Protects Neurons from Glutamate-Induced Excitotoxicity by Membrane Anchored AKAP79-PKA Interaction Network

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Kui Chen ◽  
Yu An ◽  
Lu Tie ◽  
Yan Pan ◽  
Xuejun Li
Keyword(s):  
Ampa Receptor ◽  
Drug Target ◽  
Interaction Network ◽  
Subcellular Location ◽  
Dependent Manner ◽  
Potential Drug Target ◽  
Neuroprotective Effects ◽  
Signal Relay ◽  
Anchoring Protein ◽  
Stimulation Of

Now stimulation of AMPA receptor as well as its downstream pathways is considered as potential central mediators in antidepressant mechanisms. As a signal integrator which binds to AMPA receptor, A-kinase anchoring protein 79-(AKAP79-) PKA complex is regarded as a potential drug target to exert neuroprotective effects. A well-tolerated and multitarget drug curcumin has been confirmed to exert antidepressant-like effects. To explore whether AKAP79-PKA complex is involved in curcumin-mediated antiexcitotoxicity, we detected calcium signaling, subcellular location of AKAP79-PKA complex, phosphorylation of glutamate receptor, and ERK and AKT cascades. In this study, we found that curcumin protected neurons from glutamate insult by reducing Ca2+influx and blocking the translocation of AKAP79 from cytomembrane to cytoplasm. In parallel, curcumin enhanced the phosphorylation of AMPA receptor and its downstream pathways in PKA-dependent manner. If we pretreated cells with PKA anchoring inhibitor Ht31 to disassociate PKA from AKAP79, no neuroprotective effects were observed. In conclusion, our results show that AKAP79-anchored PKA facilitated the signal relay from AMPA receptor to AKT and ERK cascades, which may be crucial for curcumin-mediated antiexcitotoxicity.

scholarly journals Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes

2019 ◽  
Vol 30 (13) ◽  
pp. 1598-1609 ◽  
Author(s):  
Erica G. Colicino ◽  
Katrina Stevens ◽  
Erin Curtis ◽  
Lindsay Rathbun ◽  
Michael Bates ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Binding Partner ◽  
Mitotic Kinase ◽  
Daughter Cells ◽  
Chromosome Distribution ◽  
Spindle Poles ◽  
Mother Centriole

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.

scholarly journals Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte.

1989 ◽  
Vol 108 (1) ◽  
pp. 127-139 ◽  
Author(s):  
E Kordeli ◽  
J Cartaud ◽  
H O Nghiêm ◽  
A Devillers-Thiéry ◽  
J P Changeux
Keyword(s):  
Plasma Membrane ◽  
Acetylcholine Receptor ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Asymmetric Distribution ◽  
Subsequent Stage ◽  
Torpedo Marmorata ◽  
Intracellular Compartments ◽  
Membrane Compartments

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

scholarly journals Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

2015 ◽  
Vol 210 (1) ◽  
pp. 45-62 ◽  
Author(s):  
Melpomeni Platani ◽  
Laura Trinkle-Mulcahy ◽  
Michael Porter ◽  
A. Arockia Jeyaprakash ◽  
William C. Earnshaw
Keyword(s):  
Mitotic Spindle ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Spindle Formation ◽  
Major Function ◽  
Mitotic Kinases ◽  
Spindle Poles

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.

scholarly journals Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2

2004 ◽  
Vol 166 (2) ◽  
pp. 167-172 ◽  
Author(s):  
Ulrike Gruneberg ◽  
Rüdiger Neef ◽  
Reiko Honda ◽  
Erich A. Nigg ◽  
Francis A. Barr
Keyword(s):  
Chromosome Segregation ◽  
Aurora B ◽  
Dual Control ◽  
Aurora B Kinase ◽  
Central Spindle ◽  
Binding Partner ◽  
Mitotic Kinases ◽  
Spatial Regulation

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B–INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.

scholarly journals The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion

2014 ◽  
Vol 289 (44) ◽  
pp. 30426-30442 ◽  
Author(s):  
Tristan Thwaites ◽  
Ana T. Nogueira ◽  
Ivan Campeotto ◽  
Ana P. Silva ◽  
Scott S. Grieshaber ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesion Kinase ◽  
Host Cell ◽  
Focal Adhesion ◽  
Quantitative Imaging ◽  
Actin Assembly ◽  
Actin Remodeling ◽  
Binding Partner ◽  
Obligate Intracellular Pathogen ◽  
Functional Relevance

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34Arc, and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

scholarly journals Molecular Distinctions between Aurora A and B: A Single Residue Change Transforms Aurora A into Correctly Localized and Functional Aurora B

2009 ◽  
Vol 20 (15) ◽  
pp. 3491-3502 ◽  
Author(s):  
Fabienne Hans ◽  
Dimitrios A. Skoufias ◽  
Stefan Dimitrov ◽  
Robert L. Margolis
Keyword(s):  
Aurora B ◽  
Aurora A ◽  
Mitotic Progression ◽  
Primary Sequence ◽  
Binding Partner ◽  
Mitotic Kinases ◽  
Reciprocal Mutation

Aurora A and Aurora B, paralogue mitotic kinases, share highly similar primary sequence. Both are important to mitotic progression, but their localizations and functions are distinct. We have combined shRNA suppression with overexpression of Aurora mutants to address the cause of the distinction between Aurora A and Aurora B. Aurora A residue glycine 198 (G198), mutated to asparagine to mimic the aligned asparagine 142 (N142) of Aurora B, causes Aurora A to bind the Aurora B binding partner INCENP but not the Aurora A binding partner TPX2. The mutant Aurora A rescues Aurora B mitotic function. We conclude that binding to INCENP is alone critical to the distinct function of Aurora B. Although G198 of Aurora A is required for TPX2 binding, N142G Aurora B retains INCENP binding and Aurora B function. Thus, although a single residue change transforms Aurora A, the reciprocal mutation of Aurora B does not create Aurora A function. An Aurora A-Δ120 N-terminal truncation construct reinforces Aurora A similarity to Aurora B, because it does not associate with centrosomes but instead associates with kinetochores.

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