21 research outputs found
DNA-protein interaction dynamics at the Lamin B2 replication origin
No full textThe regulation of human DNA replication operates via a time-defined program of
activation and deactivation of approximately 30,000 replication origins distributed
along the genome. Due to the complexity of this process, each step requires a
sequence of cascade checkpoints and licensing events, most of which are well
conserved from yeasts to humans. A multi-protein complex assembles onto each
origin causing the local unwinding of the DNA double helix and the start of two
oppositely moving replicative forks. Despite the cis-acting elements necessary for
origin firing are almost elucidated, the mechanism that governs the selection of a
specific DNA sequence as human (and, more generally, metazoan) origin, in the
course of G1 phase of the cell cycle, is still poorly understood. The lack of DNA sequence
consensus between replication origins characterized so far, together with the
poor binding-specificity displayed by the Origin Recognition Complex, suggest that
origin selection might rather be determined by local chromatin structures and/or
trans-acting factors. With regard to the latter possibility, it was interesting to find out
that a DNA region specifically bound by the AP-1 proteins, is located close to the
start site of the human Lamin B2 replication origin.
In the study conducted during this Ph.D. program, the possible role of AP-1
transcription factors in origin specification was explored by investigating the
involvement the principal moieties of this protein family, c-Fos and c-Jun, within the
replicative complexes in living human cells. The data reported in this thesis provides
evidence that both c-Fos and c-Jun interact with the LaminB2 origin of DNA
replication and indeed participates in origin function. Participation of these proteins
to origin binding is consistent with their interaction with both ORC4 and HOXC13,
two members of the replicative complex, and is cell cycle defined, occurring before
origin firing. Furthermore the observations point to the existence of specific and dynamic structural reorganizations of the complexes assembled at the origin region
along with origin activation. In this view, AP-1 proteins could contribute to recruit
and stabilize the replicative complexes onto the LaminB2 origin, in presence of
specific chromatin and topological configurations
Atomic force microscopy and lamins: a review study towards future, combined investigations
No full textIn the last decades, atomic force microscopy (AFM) underwent a rapid and stunning development,
especially for studying mechanical properties of biological samples. The numerous discoveries relying
to this approach, have increased the credit of AFM as a versatile tool, and potentially eligible
as a diagnostic equipment. Meanwhile, it has become strikingly evident that lamins are involved on
the onset and development of certain diseases, including cancer, Hutchinson-Gilford progeria syndrome,
cardiovascular pathologies, and muscular dystrophy. A new category of pathologies has
been defined, the laminopathies, which are caused by mutations in the gene encoding for A-type
lamins. As the majority of medical issues, lamins, and all their related aspects can be considered as
a quite complex problem. Indeed, there are many facets to explore, and this definitely requires a
multidisciplinary approach. One of the most intriguing aspects concerning lamins is their remarkable
contribute to cells mechanics. Over the years, this has led to the speculation of the so-called
“structural hypothesis”, which attempts to elucidate the etiology and some features of the laminopathies.
Among the various techniques tried to figure out the role of lamins in the cells mechanics,
the AFM has been already successfully applied, proving its versatility. Therefore, the present work
aims both to highlight the qualities of AFM and to review the most relevant knowledge about lamins,
in order to promote the study of the latter, taking advantage from the forme
DNA-protein interaction dynamics at the Lamin B2 replication origin
No full textTo date, a complete understanding of the molecular events leading to DNA replication origin activation in mammalian cells still remains elusive. In this work, we report the results of a high resolution chromatin immunoprecipitation study to detect proteins interacting with the human Lamin B2 replication origin. In addition to the pre-RC component ORC4 and to the transcription factors USF and HOXC13, we found that 2 components of the AP-1 transcription factor, c-Fos and c-Jun, are also associated with the origin DNA during the late G1 phase of the cell cycle and that these factors interact with ORC4. Both DNA replication and AP-1 factor binding to the origin region were perturbed by cell treatment with merbarone, a topoisomerase II inhibitor, suggesting that DNA topology is essential for determining origin function
Easy fabrication of aligned PLLA nanofibers-based 2D scaffolds suitable for cell contact guidance studies
No full textAn easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(L-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns,with fiber widths between 40 and 500 nmand lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and,more in general, the behavior of cells onto nanofibrous scaffolds), at very lowcosts and in an extremely easy way, accessible to virtually any laboratory
An engineering insight into the relationship of selective cytoskeletal impairment and biomechanics of HeLa cells
No full textIt is widely accepted that the pathological state of cells is characterized by a modification of mechanical properties, affecting cellular shape and viscoelasticity as well as adhesion behaviour and motility. Thus, assessing these parameters could represent an interesting tool to monitor disease development and progression, but also the effects of drug treatments. Since biomechanical properties of cells are strongly related to cytoskeletal architecture, in this work we extensively studied the effects of selective impairments of actin microfilaments and microtubules on HeLa cells through force-deformation curves and stress relaxation tests with atomic force microscopy. Confocal microscopy was also used to display the effects of the used drugs on the cytoskeletal structure. In synergy with the aforementioned methods, stress relaxation data were used to assess the storage and loss moduli, as a complementary way to describe the influence of cytoskeletal components on cellular viscoelasticity. Our results indicate that F-actin and microtubules play a complementary role in the cell stiffness and viscoelasticity, and both are fundamental for the adhesion properties. Our data support also the application of biomechanics as a tool to study diseases and their treatments
Cellular biomechanics impairment in keratinocytes is associated with a C-terminal truncated desmoplakin: An atomic force microscopy investigation
No full textIn a tissue continuously challenged by mechanical stresses, such as the skin or the heart, cells perceive information about their microenvironment through several adhesive protein complexes and activate cell-signaling events to maintain tissue cohesion. Consequently, alteration of cell adhesion components leads to aberrant assembly of the associated cytoplasmic scaffolding and signaling pathways, which may reflect changes to the tissue physiology and mechanical resistance. Desmoplakin is an essential component of the cell-cell junction, anchoring the desmosomal protein complex to the intermediate filaments (IFs). Inherited mutations in desmoplakin are associated with both heart and skin disease (cardiocutaneous syndrome). In this study, we investigated the mechanical properties of human keratinocytes harboring a cardiocutaneous-associated homozygous C-terminal truncation in desmoplakin (JD-1) compared to a control keratinocyte line (K1). Using Single Cell Force Spectroscopy (SCFS) AFM-based measurements, JD-1 keratinocytes displayed an overall alteration in morphology, elasticity, adhesion capabilities and viscoelastic properties, highlighting the profound interconnection between the adhesome pathways and the IF scaffold
Knock Down of Plakophillin 2 Dysregulates Adhesion Pathway through Upregulation of miR200b and Alters the Mechanical Properties in Cardiac Cells
No full textAbstract: Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte–myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM
The Cardiomyopathy Lamin A/C D192G Mutation Disrupts Whole-Cell Biomechanics in Cardiomyocytes as Measured by Atomic Force Microscopy Loading-Unloading Curve Analysis
No full textAtomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties
Biomechanical defects and rescue of cardiomyocytes expressing pathologic nuclear lamins
No full textGiven the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM) and molecular modeling was used to analyze the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte (NRVM) model
AFM single-cell force spectroscopy links altered nuclear and cytoskeletal mechanics to defective cell adhesion in cardiac myocytes with a nuclear lamin mutation
No full textPrevious investigations suggested that lamin A/C gene (LMNA) mutations, which cause a variety of human diseases including muscular dystrophies and cardiomyopathies, alter the nuclear mechanical properties. We hypothesized that biomechanical changes may extend beyond the nucleus.
Combining atomic force microscopy (AFM), molecular and cellular biology, we studied the biomechanical properties of cardiomyocytes expressing the cardiomyopathy LMNA D192G mutation, and attempted rescue through the subsequent introduction of wild-type LMNA.
Neonatal rat ventricular myocytes (NRVMs) were infected with adenoviral vectors carrying either human LMNA wild-type or D192G gene. LMNA protein expression was confirmed up to day 6 by western blot. Live-cell AFM force-deformation curves from day 1 through day 6 showed that the nuclei of NRVMs expressing LMNA D192G displayed increased stiffness compared to both uninfected and wild-type expressing cells, with a peak at 48 hours (3-fold increase in nuclear Young modulus, p<0.0001). Furthermore, mutant NRVMs showed a significant reduction in the adhesion area between AFM probe and cell membrane, impaired cytoskeletal deformation measured by relaxation force test, associated with alteration of the cytoskeletal actin network by confocal microscopy. The altered actin network and mechanical properties of LMNA D192G NRVMs were rescued by the subsequent expression of wild-type LMNA.
In conclusion, mutant LMNA deleterious effects appear to extend beyond the increased nuclear stiffness, to include altered cytoskeletal mechanics and defective cell membrane adhesion work, observations that are likely to underpin the changes in cardiac function that characterize this severe cardiomyopathy. Finally, expression of wild-type LMNA restores the mechanical properties of mutant NRVMs
