191 research outputs found
Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity
Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth and gravitropic response, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants
Emission spectra profiling of fluorescent proteins in living plant cells
Background: Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems.
Results: Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C- terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView (TM) FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells.
Conclusions: Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists
Functional modules in the Arabidopsis core cell cycle binary protein-protein interaction network
As in other eukaryotes, cell division in plants is highly conserved and regulated by cyclin-dependent kinases (CDKs) that are themselves predominantly regulated at the posttranscriptional level by their association with proteins such as cyclins. Although over the last years the knowledge of the plant cell cycle has considerably increased, little is known on the assembly and regulation of the different CDK complexes. To map protein-protein interactions between core cell cycle proteins of Arabidopsis thaliana, a binary protein-protein interactome network was generated using two complementary high-throughput interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. Pairwise interactions among 58 core cell cycle proteins were tested, resulting in 357 interactions, of which 293 have not been reported before. Integration of the binary interaction results with cell cycle phase-dependent expression information and localization data allowed the construction of a dynamic interaction network. The obtained interaction map constitutes a framework for further in-depth analysis of the cell cycle machinery
Effect of diuresis on bother related to lower urinary tract symptoms
Aims: To explore and objectify the impact of diuresis on the presence of daytime and nighttime LUTS, and associated bother.
Methods: Participants (healthy volunteers and patients referred to our hospital for a urologic consultation) were asked to complete a 72-hour bladder diary and also had to fill in a Likert scale (0: no bother; 10: maximal bother) to evaluate the LUTS-associated bother during daytime and nighttime. Three groups were defined: no/mild bother (Bother = 7). Questionnaires in order to assess LUTS (ICIQ MLUTS and ICIQ FLUTS), quality of life (SF-36, NqOL), and quality of sleep (PSQI) were completed.
Results: During daytime and nighttime, the study of participants with a strong bother associated with LUTS revealed a statistically significant higher fluid intake than those without bother (1640 mL vs 1800 mL during daytime, P = 0.007). Also, those with higher diuresis rate had more bother related to LUTS than the others (71.3 mL/h in group 1 vs 87.3 mL/h in group 3 (P < 0.001) during daytime). As might be expected, lower urinary tract such as bladder, for example, studied through maximal voided volume does play a role. The lower the maximal voided volume is, the more bother the participants described.
Conclusions: This study pointed out the implication of diuresis in bother related to LUTS. Our findings showed that there was an increase in bother by 1 when diuresis increased by 100 mL/h
Cell cycle-dependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells
SummaryEukaryotic cells have developed different mechanisms to establish the division plane [1]. In plants, the position is determined before the onset of mitosis by the preprophase band (PPB) [2, 3]. This ring of microtubules surrounds the nucleus and disappears completely by prometaphase. An unknown marker is left behind by the PPB, providing the necessary spatial cues during cytokinesis. At the position of the PPB, cortical actin is removed or modified to generate an actin-depleted zone that was proposed to provide the structural means for phragmoplast guidance [4–6]. Here, we identify a plasma membrane domain that emerges at the onset of mitosis and persists until the end of cytokinesis. The narrow band in the plasma membrane corresponds to the position of the PPB and is prevented from accumulation of a GFP-tagged kinesin GFP-KCA1; hence, it is called the KCA-depleted zone (KDZ). The KDZ demarcates the cortical division site independent from the mitotic cytoskeleton. Cell divisions in the absence of a KDZ resulted in misplaced cell plates, suggesting that the PPB transmits a signal to the plasma membrane required for correct cell plate guidance and vesicular targeting to the cortical division site
Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity
Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants
Results of angular-stable locked intramedullary nails in the treatment of distal tibia fractures
AbstractIntroductionIntramedullary nailing in distal tibial fracture is controversial because of a lack of stability. The present study sought to assess radiological and clinical results for a new “angular-stable” locking system in difficult indications for intramedullary nailing.Material and methodA prospective study recruited 41 patients (41 tibias) with distal tibial fracture consecutively managed using angular-stable locked intramedullary nails. Radiologic assessment comprised AP and lateral lower-limb views, taken postoperatively and through to last follow-up. The mean distance was measured between fracture and joint line. Fusion, with or without malunion, primary reduction defect, non-union and secondary displacement were recorded, as were all complications.ResultsMean follow-up was 18±5 months; 3 patients were lost to follow-up. Mean fracture distance from the joint line was 63±25mm. Fusion was achieved within 3 months in 29 cases (76%); delayed fusion in 7 patients (18%) required secondary dynamization at a mean 3 months, with favorable evolution. Revision surgery was required in 2 cases: 1 for secondary displacement exceeding 10°, and 1 for non-union at 7 months. Other complications mainly comprised 4 malunions of less than 10° due to primary reduction defect.ConclusionAngular-stable locked lower-limb intramedullary nailing provided a very satisfactory fusion rate, with few complications. It is, however, a demanding procedure, especially as regards fracture reduction and nail positioning in the distal fragment.Prospective cohort studylevel IV
Lesion‐directed screening to optimize skin cancer detection in dermatology practice : an observational study
Background Early detection of skin cancer is still a major challenge in dermatology practice today. While surveillance programs are offered to high-risk patients, systematic total-body examination (TBE) in the general population is not cost-effective. In the past, we demonstrated that a lesion-directed screening (LDS) in the general population delivered similar detection rates to TBE and was less time-consuming.
Objectives To study whether a lesion-directed early-access consultation can optimize skin cancer detection in dermatology practice.
Methods In this observational study, we offered an early-access consultation in patients contacting the dermatology department concerning 1 or 2 lesions of concern meeting predefined criteria.
Results 342 persons were seen at the dermatology department after triage by phone. Skin cancer detection rate was 13.2% (4.1% for melanoma). If advised/referred by a doctor skin cancer detection rate was 23.6% (9% for melanoma). With a history of skin cancer, detection rate was 24.3% (4.3% for melanoma). In patients with no referral and a negative history of skin cancer, detection rate was 7.7% (1.7% for melanoma), which is at least triple the rates reported by population-based screening programs. In patients in whom the index lesion was benign, worry of having skin cancer had decreased significantly by the end of the consultation. Additional total-body examination in these patients had low additional detection rate (0.5%) and a high number of unnecessary excisions (number needed to excise 13).
Conclusions An early-access dermatology consultation for LDS after triage by phone resulted in high overall skin cancer and melanoma detection rates. Our data indicate that performing TBE is especially useful if the index lesion is suspicious. In addition to surveillance programs in high-risk patients, LDS may be a way to optimize skin cancer detection in the general population and use available time more efficiently in daily dermatology practice
Rapamycin-dependent delocalization as a novel tool to reveal protein-protein interactions in plants
Identifying protein-protein interactions (PPI) is crucial to understand any type of biological process. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only few PPI tools allow visualizing higher order interactions. Here, we present a novel and conditional in vivo PPI tool for plant research. Knocksideways in plants (KSP) uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations, which other PPI systems hold. It is an in vivo tool, it is flexible concerning the orientation of protein tagging as long as this does not interfere with the interaction and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore does not require additional controls. The interactions can be quantified and in high throughput by the scripts that we provide. Finally, we demonstrate that KSP can visualize higher-order interactions. It is therefore a versatile tool, complementing the PPI methods field with unique characteristics and applications
High temporal resolution reveals simultaneous plasma membrane recruitment of TPLATE complex subunits
The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC in Arabidopsis (Arabidopsis thaliana) contains six evolutionarily conserved subunits and two plant-specific subunits, AtEH1/Pan1 and AtEH2/Pan1, although cytoplasmic proteins are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and lowered temperatures. Lowering the temperature slowed down endocytosis, thereby enhancing the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE and the AtEH/Pan1 proteins exhibited simultaneous recruitment at the PM. These results, together with co-localization analysis of different TPC subunits, allow us to conclude that TPC in plant cells is not recruited to the PM sequentially but as an octameric complex
- …