EXO70C2 is a key regulatory factor for optimal tip growth of pollen

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes

Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

Functions of the exocyst complex in secretion and cell wall biogenesis

The mechanical strength of plant tissues and organs can be attributed to specific properties of the cell wall. In many cases, in order to establish their final shape, cells deposit various cell wall materials in a localized manner. This is achieved by highly organized action of the endomembrane system which is essential for biosynthesis and secretion of cell wall proteins and polysaccharides. The exocyst complex is a conserved tethering complex in eukaryotes and it is involved in tethering of secretory vesicles to the sites of secretion at the plasma membrane. In this study, we address several aspects of the plant exocyst complex architecture and cell wall development using molecular biology techniques and advanced confocal microscopy. We demonstrated that two SEC10 exocyst subunits are present in Arabidopsis thaliana and share redundant functions. We also showed that the architecture of the plant exocyst complex shares several structural features with the yeast one. We demonstrated the importance of the functional EXO84b exocyst subunit for normal tracheary element development and showed that the main constituents of the secondary cell walls are deposited normally in exocyst mutants. We described a clear difference in the exocyst microtubule-independent dynamics in epidermal cells vs. cell type..

Funkce komplexu exocyst v sekreci a biogenezi buněčné stěny

SOUHRN Mechanická pevnost rostlinných pletiv a orgánů může být přičítána specifickým vlastnostem buněčné stěny. V mnoha případech se rozličné materiály buněčné stěny ukládají v buňkách lokalizovaným způsobem, za účelem dosažení konečného tvaru buňky. Toto zajišťuje vysoce organizované působení endomembránového systému, který je nezbytný pro biosyntézu a sekreci proteinů a polysacharidů tvořících buněčnou stěnu. U eukaryot je exocyst evolučně konzervovaný poutací komplex, který přichycuje sekreční váčky v místech sekrece na plazmatické membráně. V této práci jsme řešili několik aspektů stavby rostlinného komplexu exocyst a tvorby buněčné stěny za použití technik molekulární biologie a pokročilé konfokální mikroskopie. Ukázali jsme, že v Arabidopsis thaliana jsou přítomny dvě podjednotky exocystu SEC10, jejichž funkce jsou vzájemně zastupitelné. Také jsme prokázali, že uspořádání rostlinného komplexu exocyst sdílí strukturální rysy exocystu obdobně jako u kvasinek. Zaznamenali jsme význam funkční podjednotky exocystu EXO84b pro normální vývoj vodivých pletiv, a zjistili, že u mutantů v podjednotkách exocystu se hlavní složky sekundární buněčné stěny ukládají normálně. Popsali jsme rozdílnou dynamiku exocystu v epidermálních buňkách, která je nezávislá na mikrotubulech, narozdíl od buněk vyvíjejících se v...The mechanical strength of plant tissues and organs can be attributed to specific properties of the cell wall. In many cases, in order to establish their final shape, cells deposit various cell wall materials in a localized manner. This is achieved by highly organized action of the endomembrane system which is essential for biosynthesis and secretion of cell wall proteins and polysaccharides. The exocyst complex is a conserved tethering complex in eukaryotes and it is involved in tethering of secretory vesicles to the sites of secretion at the plasma membrane. In this study, we address several aspects of the plant exocyst complex architecture and cell wall development using molecular biology techniques and advanced confocal microscopy. We demonstrated that two SEC10 exocyst subunits are present in Arabidopsis thaliana and share redundant functions. We also showed that the architecture of the plant exocyst complex shares several structural features with the yeast one. We demonstrated the importance of the functional EXO84b exocyst subunit for normal tracheary element development and showed that the main constituents of the secondary cell walls are deposited normally in exocyst mutants. We described a clear difference in the exocyst microtubule-independent dynamics in epidermal cells vs. cell type...Katedra experimentální biologie rostlinDepartment of Experimental Plant BiologyPřírodovědecká fakultaFaculty of Scienc

Cracking the green wall code: insights into cell wall integrity across organisms

Cell walls are not just passive barriers; they are dynamic and adaptable structures that are actively remodeled in response to both internal and external cues. They are crucial in defining cellular identity, ensuring structural integrity, and mediating interactions with the environment. The concept of cell wall integrity (CWI) encompasses the mechanisms by which cells monitor and maintain their walls, ensuring proper function and response to challenges. While significant knowledge has been accumulated on CWI in certain model organisms, there remains a vast landscape of uncharted territory in others. In this review, we aim to bridge this gap, offering a comparative perspective on CWI across different evolutionary lineages, from the well-studied yeasts to the diverse world of plants. We focus especially on the green lineage –the group of green algae and land plants, hence the green wall–, but also consider some insights from organisms with radically different lifestyles and cell wall arrangements, which serves as a base to some intriguing questions about the role of CWI across evolution and environmental adaptation

Chiari pelvic osteotomy in treatment of hip dysplasia

Chiari pelvic osteotomy is a surgical procedure having been performed for almost sixty years in patients with the insuffcient coverage of the femoral head. It is most frequently used in young patients with dysplastic acetabular sockets as a part of developmental hip dysplasia. Even though performance of the Chiari osteotomy is associated with positive therapeutical results, above all, its main goal is to delay inevitable degenerative changes. Original surgical technique has been modified and improved over time. Nevertheless, the basic idea has remained unchanged - increasing of the femoral head coverage by medial displacement of the distal part of the pelvis along with capsular interpositioning. Given the complexity of operation, the complication percentage is rather low. Chiari pelvic osteotomy has lost its actuality and importance during this past six decades. The role of Chiari pelvic osteotomy has been considerably taken over by other more efficient and more lasting surgical procedures. Nonetheless, Chiari pelvic osteotomy is still present in modern orthopedic practice, above all as „salvage“ osteotomy. [Projekat Ministarstva nauke Republike Srbije, br. 41004

Diagnosis and treatment of chondroblastoma: Our experience

The paper presented the results of diagnostics and treatment of patients with diagnosis of chondroblastoma, treated at the Institute of Orthopedic Surgery "Banjica", Belgrade. A total of 30 patients were analyzed, involving the period from 1975-2004. All important data were obtained using the complete medical documentation, physical examination, radiographic findings and available additional diagnostic procedures. The proximal part of tibia, the proximal part of humerus and the distal part of femur were the most common sites of tumor, accounting for 63% of cases. Higher incidence of chondroblastoma was found in male patients, especially in the second decade of life. Pathohistological tumor verification was done in all patients. The patients were then treated by different surgical procedures, both on account of primary lesion and recurrence. Curettage and osteoplasty using the auto- and homograft were carried out in 21 patients, wide resection in nine cases, and amputation was performed in three cases. One patient had radiotherapy due to recurrence of lesion, nevertheless malignant transformation of chondroblastoma occurred in time. Seven patients manifested local recurrence, and one of them even had relapse on two occasions and malignant transformation of chondroblastoma. Malignant lesions were found in three cases (10% of patients); one lesion was diagnosed as primary while other two malignancies were detected only after recurrence, and the treatment was completed by lower leg amputation

Total hip arthroplasty

Total hip arthroplasty is most common reconstructive hip procedure in adults. In this surgery we replace some parts of the upper femur and acetabulum with biocompatible materials. The main goal of this surgery is to eliminate pain and regain full extent of joint motion, maintaining hip stability. Surgical technique, biomaterials, design of the prosthesis and fixation techniques have evolved with time adjusting to each other. After total hip arthroplasty patients’ quality of life should be improved. There are many various postoperative complications. Some of them are fatal, and some are minor, which may become manifested years after surgery. Each next surgical procedure following previous hip surgery is associated with considerably lower chances to be successful. Therefore, in primary total hip arthroplasty, preoperative evaluation and preparation of patients are essential. Every orthopaedic surgeon needs to improve already adopted surgical skills applying them with precision and without compromise, with the main goal to achieve long-term durability of the selected implant. The number of total hip arthroplasties will also increase in future, and newer and higher quality materials will be used

Treatment of missed Monteggia lesion in children: Case report

Introduction. A Monteggia lesion is a dislocation of the radial head associated with a fracture with the proximal third of the ulna. It is rare in children and the dislocation of the radial head is often missed at the time of injury. There are a lot of described treatment methods: open reduction of the radial head and reconstruction of the annular ligament combined with ulnar osteotomy, the same method without reconstruction of the annular ligament, gradual lengthening and angulation of the ulna by Ilizarov method without the opening of radiocapitelar joint. Case Outline. A 14-year-old boy had been diagnosed with Monteggia lesion type Bado II three years before the admission to hospital. Previously nonoperatively treated, the missed radial head dislocation Bi-phase treatment had been done. Firstly, the distraction Ilizarov device was placed on the forearm, corticotomy of the ulna was done, distraction lasted fifteen days. Secondly, after achieving 1.5 cm of new bone and good level of the radial head, a reduction mechanism was incorporated into the Ilizarov device. The radiocapitelar joint was not opened, the reconstruction of the annular ligament was not done. After the radial head reduction and new bone maturation (1.5 months), the Ilizarov device was taken off. Conclusion. The described method of treatment has several important advantages: radial head reduction may be done without the joint opening, the recovery is very fast and easy after that; the elbow and forearm appear cosmetically very well after the operation; the treatment process is short, the arm is in use all the time, the absence from school is not needed, the achieved result is permanent

SUMMARY

Introduction Insufficient femoral head coverage is found in a variety of diseases, with acetabular dysplasia as the most frequent disorder and triple pelvic osteotomy as the most recently introduced surgical treatment. Objective This study analyses pre- and postoperative pathoanatomic characteristics of triple in comparison to Salter and Chiari osteotomies, with a logistic regression analysis of outcome predictor and effect explanator factors in relation to the chosen type of operation