scholarly journals Subcellular drug targeting illuminates local kinase action

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Paula J Bucko ◽  
Chloe K Lombard ◽  
Lindsay Rathbun ◽  
Irvin Garcia ◽  
Akansha Bhat ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Super Resolution ◽  
Biological Processes ◽  
Anchoring Proteins ◽  
Intracellular Compartments ◽  
Mitotic Spindle Assembly ◽  
Targeted Inhibition ◽  
Super Resolution Microscopy

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

scholarly journals P176Distribution of endotheltial surface layer anchoring proteins analyzed with super resolution microscopy

2014 ◽  
Vol 103 (suppl 1) ◽  
pp. S31.2-S31
Author(s):  
A Marki ◽  
M Boehme ◽  
S Sigrist ◽  
A Zakrzewicz ◽  
A Pries
Keyword(s):  
Surface Layer ◽  
Super Resolution ◽  
Anchoring Proteins ◽  
Super Resolution Microscopy

scholarly journals FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

2020 ◽  
Author(s):  
Eleonora Turco ◽  
Irmgard Fischer ◽  
Sascha Martens
Keyword(s):  
Kinase Activity ◽  
De Novo ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Degradation Pathway ◽  
Autophagosome Formation ◽  
Ubiquitinated Proteins ◽  
The Many ◽  
Super Resolution Microscopy ◽  
Insight Into

AbstractMacroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.

scholarly journals Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

2020 ◽  
Author(s):  
Ryu J. Iwatate ◽  
Akira Yoshinari ◽  
Noriyoshi Yagi ◽  
Marek Grzybowski ◽  
Hiroaki Ogasawara ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Super Resolution ◽  
Cell Plate ◽  
Protein Labeling ◽  
Synthetic Dyes ◽  
Biological Processes ◽  
Major Drawback ◽  
Specific Proteins ◽  
Super Resolution Microscopy

AbstractSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

scholarly journals Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

2021 ◽  
Author(s):  
Arunima Sakunthala ◽  
Debalina Datta ◽  
Ambuja Navalkar ◽  
Laxmikant Gadhe ◽  
Pradeep Kadu ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Super Resolution ◽  
Cellular Internalization ◽  
Biological Processes ◽  
Secondary Nucleation ◽  
Size Dependent ◽  
Distinct Mechanism ◽  
In Vitro Reconstitution ◽  
Super Resolution Microscopy

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

scholarly journals The mitotic spindle is chiral due to torques generated by motor proteins

2017 ◽  
Author(s):  
Maja Novak ◽  
Bruno Polak ◽  
Juraj Simunić ◽  
Zvonimir Boban ◽  
Barbara Kuzmić ◽  
...  
Keyword(s):  
Theoretical Model ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Metaphase Plate ◽  
Super Resolution ◽  
Left Handed ◽  
Associated Proteins ◽  
Balance Of Forces ◽  
Micro Machine ◽  
Super Resolution Microscopy

AbstractMitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins1,2. Forces are required for the congression of chromosomes to the metaphase plate and separation of chromatids in anaphase3-6. However, torques may also exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. The average helicity of the bundles with respect to the spindle axis is 1.2°/μm. Inactivation of kinesin-5 (Eg5/Kif11) abolished the chirality of the spindle, suggesting that this motor generates the helical shape of microtubule bundles. To explain the observed shapes, we introduce a theoretical model for the balance of forces and torques acting in the spindle, and show that torque is required to generate the helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its architecture.

scholarly journals Affimer proteins are versatile and renewable affinity reagents

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Christian Tiede ◽  
Robert Bedford ◽  
Sophie J Heseltine ◽  
Gina Smith ◽  
Imeshi Wijetunga ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Super Resolution ◽  
Biological Processes ◽  
Affinity Reagents ◽  
Tumour Antigens ◽  
Target Molecules ◽  
Super Resolution Microscopy

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Visualizing the ‘backbone’ of focal adhesions

2018 ◽  
Vol 2 (5) ◽  
pp. 677-680 ◽  
Author(s):  
Samuel F. H. Barnett ◽  
Pakorn Kanchanawong
Keyword(s):  
Protein Engineering ◽  
Structural Organization ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Biological Processes ◽  
Recent Application ◽  
Super Resolution Microscopy

To understand how complex machines perform their functions, it is essential to map out the ‘blueprints’ of how their internal components are organized. Focal adhesions (FAs) are complex mechanobiological structures involved in a plethora of cell biological processes. The application of super-resolution microscopy in concert with protein engineering offers one approach to unravel the complexity of how individual proteins are organized within FAs. In our recent application, the FA protein talin was found to form a direct structural and physical link between integrin and actin. Interestingly, engineered talin constructs with alternate lengths rescaled the FA nanostructure accordingly. This helped establish that talin could be analogous to the backbone of FAs, serving as the mechanosensitive master coordinator of FA structural organization.

scholarly journals Compartimentalized control of Cdk1 drives mitotic spindle assembly

2021 ◽  
Author(s):  
Angela Flavia Serpico ◽  
Francesco Febbraro ◽  
Caterina Pisauro ◽  
Domenico Grieco
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Growth Promoting ◽  
Fraction Bound

During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark mitosis onset leading to interphase cytoskeleton dissolution and mitotic spindle assembly. Once activated by Cdc25, that reverses inhibitory phosphorylation operated by Wee1/Myt1, Cdk1 clears the cytoplasm from microtubules by inhibiting microtubule associated proteins (MAPs) with microtubule growth-promoting properties. Nevertheless, some of these MAPs are required for spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escaped Cdc25 action and remained inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibited or promoted spindle assembly, respectively. Furthermore, polymerizing spindle microtubules fostered i-Cdk1 by aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartimentalized control of Cdk1 activity.

scholarly journals Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis

2017 ◽  
Author(s):  
Liang Ge ◽  
Min Zhang ◽  
Samuel J Kenny ◽  
Dawei Liu ◽  
Miharu Maeda ◽  
...  
Keyword(s):  
Super Resolution ◽  
Membrane Vesicles ◽  
Membrane Component ◽  
Autophagosome Formation ◽  
Er Exit Sites ◽  
Intracellular Compartments ◽  
A Subunit ◽  
Intermediate Compartment ◽  
Copii Vesicles ◽  
Super Resolution Microscopy

AbstractAutophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy
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