scholarly journals A molecular mechanism for the procentriole recruitment of Ana2

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Sarah K. Speed ◽  
John M. Ryniawec ◽  
Daniel W. Buster ◽  
Carey J. Fagerstrom ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Kinase Activity ◽  
Outer Wall ◽  
Single Site ◽  
Structural Motif ◽  
Centriole Duplication ◽  
Short Region ◽  
N Terminus ◽  
Mother Centriole ◽  
Daughter Centriole

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2–G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.

scholarly journals A molecular mechanism for the procentriole recruitment of Ana2

2019 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Sarah K. Speed ◽  
Daniel W. Buster ◽  
Carey J. Fagerstrom ◽  
Brian J. Galletta ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Kinase Activity ◽  
Surface Protein ◽  
Single Site ◽  
Centriole Duplication ◽  
C Terminus ◽  
N Terminus ◽  
Mother Centriole ◽  
Centriole Assembly ◽  
Daughter Centriole

AbstractCentriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STIL’s C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2’s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2’s N-terminus which increases Ana2’s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

scholarly journals Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication

2010 ◽  
Vol 21 (4) ◽  
pp. 547-561 ◽  
Author(s):  
James E. Sillibourne ◽  
Frederik Tack ◽  
Nele Vloemans ◽  
An Boeckx ◽  
Sathiesan Thambirajah ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Activity ◽  
Proteasome Inhibition ◽  
Centrosome Duplication ◽  
Active Fraction ◽  
Kinase Activation ◽  
Centriole Duplication ◽  
M Phase ◽  
Mother Centriole ◽  
Daughter Centriole

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.

scholarly journals Centrobin–tubulin interaction is required for centriole elongation and stability

2011 ◽  
Vol 193 (4) ◽  
pp. 711-725 ◽  
Author(s):  
Radhika Gudi ◽  
Chaozhong Zou ◽  
Jun Li ◽  
Qingshen Gao
Keyword(s):  
Molecular Mechanism ◽  
Early Stage ◽  
Centrosome Duplication ◽  
Centriole Duplication ◽  
The Stability ◽  
Daughter Centriole

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.

scholarly journals Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state

2018 ◽  
Vol 29 (23) ◽  
pp. 2874-2886 ◽  
Author(s):  
Cody J. Boese ◽  
Jonathan Nye ◽  
Daniel W. Buster ◽  
Tiffany A. McLamarrah ◽  
Amy E. Byrnes ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Dominant Role ◽  
Kinase Domain ◽  
Feedback Mechanism ◽  
Phosphorylation State ◽  
Negative Feedback Mechanism ◽  
Centriole Duplication ◽  
C Terminus ◽  
N Terminus ◽  
Opposing Effects

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

scholarly journals Self-organization of Plk4 regulates symmetry breaking in centriole duplication

2018 ◽  
Author(s):  
Shohei Yamamoto ◽  
Daiju Kitagawa
Keyword(s):  
Symmetry Breaking ◽  
Self Assembly ◽  
Self Organization ◽  
Spatial Patterning ◽  
Centriole Duplication ◽  
Dissociation Dynamics ◽  
Mother Centriole ◽  
The Asymmetry ◽  
Single Focus ◽  
Daughter Centriole

AbstractDuring centriole duplication, a single daughter centriole is formed near the mother centriole. The mechanism that determines a single duplication site is unknown. Here, we demonstrate that intrinsic self-organization of Plk4 underlies symmetry breaking in centriole duplication. We show that in its nonphosphorylated state, Plk4 preferentially self-assembles via a disordered linker and that this self-assembly is prevented by autophosphorylation. Consistently, the dissociation dynamics of centriolar Plk4 are controlled by autophosphorylation. We further found that autophophorylated Plk4 is localized as a single focus around the mother centriole before procentriole formation, and is subsequently targeted for STIL-HsSAS6 loading. Perturbing Plk4 self-organization affects the asymmetry of centriolar Plk4 distribution and centriole duplication. We propose that the spatial patterning of Plk4 directs a single duplication site per mother centriole.

scholarly journals Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

2021 ◽  
Author(s):  
Neil Henry James Cunningham ◽  
Imene Bouhlel ◽  
Paul Thomas Conduit
Keyword(s):  
Nuclear Envelope ◽  
Single Mother ◽  
S Phase ◽  
Animal Cells ◽  
Simultaneous Formation ◽  
Centriole Duplication ◽  
External Cues ◽  
Mother Centriole ◽  
Pericentriolar Material ◽  
Daughter Centriole

Centrosomes are important organisers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material (PCM), which nucleates and organises the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new daughter centriole is built orthogonally on one side of each radially symmetric mother centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.

scholarly journals Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

2018 ◽  
Author(s):  
Marcin Leda ◽  
Andrew J. Holland ◽  
Andrew B. Goryachev
Keyword(s):  
Symmetry Breaking ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Break Symmetry ◽  
Biophysical Model ◽  
Model Organisms ◽  
Centriole Duplication ◽  
Mother Centriole ◽  
Autocatalytic Activation

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

scholarly journals A homeostatic clock sets daughter centriole size in flies

2018 ◽  
Vol 217 (4) ◽  
pp. 1233-1248 ◽  
Author(s):  
Mustafa G. Aydogan ◽  
Alan Wainman ◽  
Saroj Saurya ◽  
Thomas L. Steinacker ◽  
Anna Caballe ◽  
...  
Keyword(s):  
Growth Rate ◽  
Drosophila Melanogaster ◽  
Inverse Relationship ◽  
S Phase ◽  
Cell Type ◽  
Linear Rate ◽  
Mother Centriole ◽  
Daughter Centriole ◽  
Correct Size

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing “mother” centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size.

scholarly journals A Novel Bradykinin-Related Peptide, RVA-Thr6-BK, from the Skin Secretion of the Hejiang Frog; Ordorrana hejiangensis: Effects of Mammalian Isolated Smooth Muscle

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 376 ◽  
Author(s):  
Yue Wu ◽  
Daning Shi ◽  
Xiaoling Chen ◽  
Lei Wang ◽  
Yuan Ying ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Single Site ◽  
Skin Secretion ◽  
Rat Uterus ◽  
Rat Ileum ◽  
Related Peptide ◽  
Naturally Occurring ◽  
N Terminus ◽  
The Difference

A novel naturally-occurring bradykinin-related peptide (BRP) with an N-terminal extension, named RVA-Thr6-Bradykinin (RVA-Thr6-BK), was here isolated and identified from the cutaneous secretion of Odorrana hejiangensis (O. hejiangensis). Thereafter, in order to evaluate the difference in myotropic actions, a leucine site-substitution variant from Amolops wuyiensis skin secretion, RVA-Leu1, Thr6-BK, was chemically synthesized. Myotropic studies indicated that single-site arginine (R) replacement by leucine (L) at position-4 from the N-terminus, altered the action of RVA-Thr6-BK from an agonist to an antagonist of BK actions on rat ileum smooth muscle. Additionally, both BK N-terminal extended derivatives (RVA-Thr6-BK and RVA-Leu1, Thr6-BK) exerted identical myotropic actions to BK, such as increasing the frequency of contraction, contracting and relaxing the rat uterus, bladder and artery preparations, respectively.

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