Microtubules: greater than the sum of the parts

The post-genomic era has produced a variety of new investigation technologies, techniques and approaches that may offer exciting insights into many long-standing questions of scientific research. The microtubule cytoskeleton is a highly conserved system that shows a high degree of internal complexity, is known to be integral to many cell systems and functions on a fundamental level. After decades of study, much is still unknown about microtubules in vivo from the control of dynamics in living cells to their responses to environmental changes and responses to other cellular processes. In the present article, we examine some outstanding questions in the microtubule field and propose a combination of emerging interdisciplinary approaches, i.e. high-throughput functional genomics techniques, quantitative and super-resolution microscopy, and in silico modelling, that could shed light on the systemic regulation of microtubules in cells by networks of regulatory factors. We propose that such an integrative approach is key to elucidate the function of the microtubule cytoskeleton as a complete responsive integral biological system.

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Ligand Geometry Controls Adhesion formation via Integrin Clustering

10.1101/435826 ◽

2018 ◽

Cited By ~ 2

Author(s):

Rishita Changede ◽

Haogang Cai ◽

Shalom Wind ◽

Michael P. Sheetz

Keyword(s):

Adhesion Formation ◽

Focal Adhesions ◽

Super Resolution ◽

Total Width ◽

Cellular Processes ◽

Cell Matrix ◽

Integrin Clustering ◽

Super Resolution Microscopy ◽

Fiber Mesh

AbstractIntegrin-mediated cell matrix adhesions are key to sensing the geometry and rigidity of the extracellular environment to regulate vital cellular processes. In vivo, the extracellular matrix (ECM) is composed of a fibrous mesh. To understand the geometry that supports adhesion formation on fibrous substrates, we patterned 10 nm gold-palladium single lines or pairs of lines (total width within 100 nm), mimicking thin single ECM fibers or a minimal mesh geometry, respectively and functionalized it with integrin binding ligand Arg-Gly-Asp (RGD). Single lines showed reduced focal adhesion kinase (FAK) recruitment and did not support cell spreading or formation of focal adhesions, despite the presence of a high density of integrin-binding ligands. Using super resolution microscopy, we observed transient integrin clusters on single lines, whereas stable 110 nm integrin clusters formed on pairs of lines similar to those on continuous substrates. This indicated that two-dimensional ligand geometry is required for adhesion formation on rigid substrates. A mechanism to form modular 100nm integrin clusters bridging the minimal fiber mesh would require unliganded integrins. We observed that integrin mutants unable to bind ligand co-clustered with ligand-bound integrins when present in an active extended conformation. Thus, these results indicate that functional integrin clusters are required to form focal adhesions and unliganded integrins can co-cluster to bridge between thin matrix fibers and can form stable integrin adhesions on dense fibrous networks.

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Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus

10.1101/2021.05.06.442895 ◽

2021 ◽

Author(s):

Markus Huemer ◽

Srikanth Mairpady Shambat ◽

Sandro Pereira ◽

Lies Van Gestel ◽

Judith Bergada-Pijuan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Ribosomal Proteins ◽

Environmental Changes ◽

Acid Stress ◽

Elongation Factor ◽

Growth Delay ◽

Lag Phase ◽

Cellular Processes ◽

Translational Activity

Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence.

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In Vivo Imaging with Microsphere-Based Super-Resolution Microscopy

2018 International Conference on Optical MEMS and Nanophotonics (OMN) ◽

10.1109/omn.2018.8454639 ◽

2018 ◽

Author(s):

Gergely Huszka ◽

Roger Krenger ◽

Martin A. M. Gijs

Keyword(s):

In Vivo Imaging ◽

Super Resolution ◽

Super Resolution Microscopy

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Decoration of myocellular lipid droplets with perilipins as a marker for in vivo lipid droplet dynamics: A super-resolution microscopy study in trained athletes and insulin resistant individuals

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/j.bbalip.2020.158852 ◽

2021 ◽

Vol 1866 (2) ◽

pp. 158852

Author(s):

Anne Gemmink ◽

Sabine Daemen ◽

Bram Brouwers ◽

Joris Hoeks ◽

Gert Schaart ◽

...

Keyword(s):

Lipid Droplet ◽

Lipid Droplets ◽

Super Resolution ◽

Microscopy Study ◽

Droplet Dynamics ◽

Insulin Resistant ◽

Super Resolution Microscopy

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Super-Resolution Microscopy of Chromatin

Genes ◽

10.3390/genes10070493 ◽

2019 ◽

Vol 10 (7) ◽

pp. 493 ◽

Cited By ~ 1

Author(s):

Birk

Keyword(s):

Gene Regulation ◽

Data Analysis ◽

Super Resolution ◽

Fluorescence Labeling ◽

Cellular Processes ◽

Direct Assessment ◽

Chromatin Interactions ◽

Resolution Imaging ◽

Super Resolution Microscopy ◽

Insight Into

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

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Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.10965-10978.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 10965-10978 ◽

Cited By ~ 39

Author(s):

Ryoichi Ono ◽

Masafumi Ihara ◽

Hideaki Nakajima ◽

Katsutoshi Ozaki ◽

Yuki Kataoka-Fujiwara ◽

...

Keyword(s):

Fusion Gene ◽

Fusion Partner ◽

Myeloproliferative Disease ◽

Cellular Processes ◽

Partner Gene ◽

Quantitative Changes ◽

High Degree ◽

Deficient Mice

ABSTRACT Septins are evolutionarily conserved GTP-binding proteins that can heteropolymerize into filaments. Recent studies have revealed that septins are involved in not only diverse normal cellular processes but also the pathogenesis of various diseases, including cancer. SEPT6 is ubiquitously expressed in tissues and one of the fusion partner genes of MLL in the 11q23 translocations implicated in acute leukemia. However, the roles of this septin in vivo remain elusive. We have developed Sept6-deficient mice that exhibited neither gross abnormalities, changes in cytokinesis, nor spontaneous malignancy. Sept6 deficiency did not cause any quantitative changes in any of the septins evaluated in this study, nor did it cause any additional changes in the Sept4-deficient mice. Even the depletion of Sept11, a close homolog of Sept6, did not affect the Sept6-null cells in vitro, thus implying a high degree of redundancy in the septin system. Furthermore, a loss of Sept6 did not alter the phenotype of myeloproliferative disease induced by MLL-SEPT6, thus suggesting that Sept6 does not function as a tumor suppressor. To our knowledge, this is the first report demonstrating that a disruption of the translocation partner gene of MLL in 11q23 translocation does not contribute to leukemogenesis by the MLL fusion gene.

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Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818824116 ◽

2019 ◽

Vol 116 (12) ◽

pp. 5533-5541 ◽

Cited By ~ 23

Author(s):

Yin-Wei Kuo ◽

Olivier Trottier ◽

Mohammed Mahamdeh ◽

Jonathon Howard

Keyword(s):

Microtubule Dynamics ◽

Dual Function ◽

Important Class ◽

Microtubule Cytoskeleton ◽

Neuronal Morphogenesis ◽

Cellular Processes ◽

Exponential Increase ◽

Net Flux ◽

Dynamic Microtubule

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases—spastin, katanin, and fidgetin—which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstitutedDrosophilaspastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin’s effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin’s in vivo phenotypes.

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Mitochondrial division, fusion and degradation

The Journal of Biochemistry ◽

10.1093/jb/mvz106 ◽

2019 ◽

Vol 167 (3) ◽

pp. 233-241 ◽

Cited By ~ 4

Author(s):

Daisuke Murata ◽

Kenta Arai ◽

Miho Iijima ◽

Hiromi Sesaki

Keyword(s):

Super Resolution ◽

Healthy Population ◽

Lipid Biochemistry ◽

Mitochondrial Division ◽

Cellular Processes ◽

Size Number ◽

Wide Range ◽

Autophagic Degradation ◽

And Function

Abstract The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

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Recent Advances in Organelle-Targeted Fluorescent Probes

Molecules ◽

10.3390/molecules26010217 ◽

2021 ◽

Vol 26 (1) ◽

pp. 217

Author(s):

Na-Eun Choi ◽

Ji-Yu Lee ◽

Eun-Chae Park ◽

Ju-Hee Lee ◽

Jiyoun Lee

Keyword(s):

Fluorescent Probes ◽

Molecular Level ◽

Imaging Techniques ◽

Super Resolution ◽

Structural Features ◽

Future Directions ◽

Cellular Processes ◽

Biochemical Pathways ◽

Recent Advances ◽

Super Resolution Microscopy

Recent advances in fluorescence imaging techniques and super-resolution microscopy have extended the applications of fluorescent probes in studying various cellular processes at the molecular level. Specifically, organelle-targeted probes have been commonly used to detect cellular metabolites and transient chemical messengers with high precision and have become invaluable tools to study biochemical pathways. Moreover, several recent studies reported various labeling strategies and novel chemical scaffolds to enhance target specificity and responsiveness. In this review, we will survey the most recent reports of organelle-targeted fluorescent probes and assess their general strategies and structural features on the basis of their target organelles. We will discuss the advantages of the currently used probes and the potential challenges in their application as well as future directions.

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