scholarly journals Three-dimensional observations of an aperiodic oscillatory gliding motility behaviour in Myxococcus xanthus using confocal interference reflection microscopy

2019 ◽  
Author(s):  
Liam M. Rooney ◽  
Lisa S. Kölln ◽  
Ross Scrimgeour ◽  
William B. Amos ◽  
Paul A. Hoskisson ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Basal Membrane ◽  
Gliding Motility ◽  
The Novel ◽  
Force Transmission ◽  
Topological Information ◽  
Type Iv ◽  
Microscopy Techniques

The Delta-proteobacterium, Myxococcus xanthus, has been used as a model for bacterial motility and to provide insights of bacterial swarming behaviours. Fluorescence microscopy techniques have shown that various mechanisms are involved in gliding motility, but these have almost entirely been limited to 2D studies and there is currently no understanding of gliding motility in a 3D context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, and we reveal aperiodic oscillatory behaviour with changes in the position of the basal membrane relative to the coverglass on the order of 90 nm in vitro. Firstly, we use a model plano-convex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus and show that cells undergo previously unobserved changes in their height as they glide. We compare the wild-type with mutants of reduced motility, which also exhibit the same changes in adhesion profile during gliding. We find that the general gliding behaviour is independent of the proton motive force-generating complex, AglRQS, and suggest that the novel behaviour we present here may be a result of recoil and force transmission along the length of the cell body following firing of the Type IV pili.

scholarly journals Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus xanthus Using Confocal Interference Reflection Microscopy

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Liam M. Rooney ◽  
Lisa S. Kölln ◽  
Ross Scrimgeour ◽  
William B. Amos ◽  
Paul A. Hoskisson ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Oscillatory Behavior ◽  
Gliding Motility ◽  
Bacterial Cells ◽  
Force Transmission ◽  
Content Type ◽  
Gliding Bacteria ◽  
Microscopy Techniques

ABSTRACT The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro. First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili. IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope.

scholarly journals Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

2021 ◽  
Author(s):  
Sofya Kuzmich ◽  
Dorota Skotnicka ◽  
Dobromir Szadkowski ◽  
Philipp Klos ◽  
María Pérez‐Burgos ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Intact Protein ◽  
Dependent Manner ◽  
Chemosensory System ◽  
Type Iv ◽  
Acetyltransferase Domain

In bacteria, the nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ-domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system, and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA and PixB act in independent pathways and have distinct functions in regulation of motility. Importance c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts acts in Frz-independent mannerto regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

scholarly journals Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus

2010 ◽  
Vol 192 (17) ◽  
pp. 4267-4274 ◽  
Author(s):  
Wesley P. Black ◽  
Florian D. Schubot ◽  
Zhuo Li ◽  
Zhaomin Yang
Keyword(s):  
Double Mutant ◽  
Kinase Activity ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Chemosensory Proteins ◽  
Downstream Target ◽  
Signaling Complex ◽  
Type Iv

ABSTRACT Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.

scholarly journals Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

2021 ◽  
Author(s):  
Sofya Kuzmich ◽  
Dorota Skotnicka ◽  
Dobromir Szadkowski ◽  
Philipp Klos ◽  
Maria Perez-Burgos ◽  
...  
Keyword(s):  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Chemosensory System ◽  
Developmental Program ◽  
Type Iv ◽  
Genes Encoding ◽  
Acetyltransferase Domain

In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility.

scholarly journals Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

Silicate-doped nano-hydroxyapatite/graphene oxide composite reinforced fibrous scaffolds for bone tissue engineering

2018 ◽  
Vol 32 (10) ◽  
pp. 1392-1405 ◽  
Author(s):  
Ali Deniz Dalgic ◽  
Ammar Z. Alshemary ◽  
Ayşen Tezcaner ◽  
Dilek Keskin ◽  
Zafer Evis
Keyword(s):  
Tissue Engineering ◽  
Graphene Oxide ◽  
Bone Tissue Engineering ◽  
Protein Adsorption ◽  
Bone Tissue ◽  
Three Dimensional ◽  
Osteosarcoma Cell ◽  
In Vitro Biocompatibility ◽  
Microscopy Techniques

In this study, novel graphene oxide–incorporated silicate-doped nano-hydroxyapatite composites were prepared and their potential use for bone tissue engineering was investigated by developing an electrospun poly(ε-caprolactone) scaffold. Nanocomposite groups were synthesized to have two different ratios of graphene oxide (2 and 4 wt%) to evaluate the effect of graphene oxide incorporation and groups with different silicate-doped nano-hydroxyapatite content was prepared to investigate optimum concentrations of both silicate-doped nano-hydroxyapatite and graphene oxide. Three-dimensional poly(ε-caprolactone) scaffolds were prepared by wet electrospinning and reinforced with silicate-doped nano-hydroxyapatite/graphene oxide nanocomposite groups to improve bone regeneration potency. Microstructural and chemical characteristics of the scaffolds were investigated by X-ray diffraction, Fourier transform infrared spectroscope and scanning electron microscopy techniques. Protein adsorption and desorption on material surfaces were studied using fetal bovine serum. Presence of graphene oxide in the scaffold, dramatically increased the protein adsorption with decreased desorption. In vitro biocompatibility studies were conducted using human osteosarcoma cell line (Saos-2). Electrospun scaffold group that was prepared with effective concentrations of silicate-doped nano-hydroxyapatite and graphene oxide particles (poly(ε-caprolactone) – 10% silicate-doped nano-hydroxyapatite – 4% graphene oxide) showed improved adhesion, spreading, proliferation and alkaline phosphatase activity compared to other scaffold groups.

scholarly journals Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis

2007 ◽  
Vol 189 (15) ◽  
pp. 5716-5727 ◽  
Author(s):  
Seetha V. Balasingham ◽  
Richard F. Collins ◽  
Reza Assalkhou ◽  
Håvard Homberset ◽  
Stephan A. Frye ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Specific Interaction ◽  
Three Dimensional ◽  
Middle Part ◽  
Dna Uptake ◽  
Type Iv ◽  
Pilus Biogenesis ◽  
Inner Membrane Protein ◽  
Mutant Phenotypes

ABSTRACT Neisseria meningitidis can be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP in trans. The pilP gene is located upstream of pilQ, and analysis of their transcripts indicated that pilP and pilQ were cotranscribed. Furthermore, analysis of the level of PilQ expression in pilP mutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect on pilQ transcription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions.

Analysis of type IV pilus and its associated motility in Myxococcus xanthus using an antibody reactive with native pilin and pili

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Yinuo Li ◽  
Renate Lux ◽  
Andrew E. Pelling ◽  
James K. Gimzewski ◽  
Wenyuan Shi
Keyword(s):  
Amino Acids ◽  
Current Model ◽  
Bacterial Species ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Soluble Form ◽  
Truncated Form ◽  
Type Iv ◽  
Oligomerization Domain

Myxococcus xanthus possesses a social gliding motility that requires type IV pili (TFP). According to the current model, M. xanthus pili attach to an external substrate and retract, pulling the cell body forward along their long axis. By analogy with the situation in other bacteria employing TFP-dependent motility, M. xanthus pili have been assumed to be composed of pilin (PilA) subunits, but this has not previously been confirmed. The first 28 amino acids of the M. xanthus PilA protein share extensive homology with the N-terminal oligomerization domain of pilins in other bacterial species. To facilitate purification, the authors engineered a truncated form of M. xanthus PilA lacking the first 28 amino acids and purified this protein in soluble form. Polyclonal antibody generated against this protein was reactive with native pilin and pili. Using this antibody, it was confirmed that TFP of M. xanthus are indeed composed of PilA, and that TFP are located unipolarly and required for social gliding motility via retraction. Using tethering as well as motility assays, details of pili function in M. xanthus social motility were further examined.

scholarly journals Microdevice for directional axodendritic connectivity between micro 3D neuronal cultures

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yixuan Ming ◽  
Md Joynal Abedin ◽  
Svetlana Tatic-Lucic ◽  
Yevgeny Berdichevsky
Keyword(s):  
Three Dimensional ◽  
Basic Feature ◽  
Neuronal Cultures ◽  
The Novel ◽  
Cortical Circuits ◽  
Cortical Cells ◽  
Neuroscience Research ◽  
Directional Connectivity ◽  
Novel Concept

AbstractNeuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (μ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an “en passant” synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.

scholarly journals Fabrication of a Polycaprolactone/Alginate Bipartite Hybrid Scaffold for Osteochondral Tissue Using a Three-Dimensional Bioprinting System

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2203 ◽  
Author(s):  
JunJie Yu ◽  
SuJeong Lee ◽  
Sunkyung Choi ◽  
Kee K. Kim ◽  
Bokyeong Ryu ◽  
...  
Keyword(s):  
Three Dimensional ◽  
The Novel ◽  
Osteochondral Defects ◽  
3D Bioprinting ◽  
Hybrid Scaffold ◽  
Osteochondral Tissue ◽  
Biological Performance ◽  
Osteochondral Tissue Engineering ◽  
Made In

Osteochondral defects, including damage to both the articular cartilage and the subchondral bone, are challenging to repair. Although many technological advancements have been made in recent years, there are technical difficulties in the engineering of cartilage and bone layers, simultaneously. Moreover, there is a great need for a valuable in vitro platform enabling the assessment of osteochondral tissues to reduce pre-operative risk. Three-dimensional (3D) bioprinting systems may be a promising approach for fabricating human tissues and organs. Here, we aimed to develop a polycaprolactone (PCL)/alginate bipartite hybrid scaffold using a multihead 3D bioprinting system. The hybrid scaffold was composed of PCL, which could improve the mechanical properties of the construct, and alginate, encapsulating progenitor cells that could differentiate into cartilage and bone. To differentiate the bipartite hybrid scaffold into osteochondral tissue, a polydimethylsiloxane coculture system for osteochondral tissue (PCSOT) was designed and developed. Based on evaluation of the biological performance of the novel hybrid scaffold, the PCL/alginate bipartite scaffold was successfully fabricated; importantly, our findings suggest that this PCSOT system may be applicable as an in vitro platform for osteochondral tissue engineering.

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