Papers by Valeria Caiolfa
Nature Cell Biology
In response to different types and intensities of mechanical force, cells modulate their physical... more In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae a...
Biology Open
Most studies addressing chromatin behaviour during preimplantation development are based on bioch... more Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.
Frontiers in Cell and Developmental Biology, 2021
HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocy... more HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocaliza...
Haematologica, 2020
Chronic Lymphocytic Leukemia (CLL) cells disseminate into supportive tissue microenvironments. To... more Chronic Lymphocytic Leukemia (CLL) cells disseminate into supportive tissue microenvironments. To investigate the mechanisms involved in leukemic cell tissue retention we developed a 3D bone marrow (BM) microenvironment that recreates CLL - BM-stromal cells interactions inside a scaffold within a bioreactor. Our system allows the parallel analysis of CLL cells retained inside the scaffold and those released in the presence/absence of pharmacological agents, mimicking tissue and circulating cell compartments, respectively. CLL cells can be retained within the scaffold only in the presence of microenvironmental elements, which through direct contact down-regulate the expression of HS1 cytoskeletal protein in CLL cells. Consist with this, the expression of HS1 was lower in CLL cells obtained from patients' BM versus CLL cells circulating in the PB. Moreover, we demonstrate that CLL cells with inactive-HS1, impaired cytoskeletal activity and a more aggressive phenotype are more like...
Journal of Biological Chemistry, 1988
We have isolated a complex of two proteins from bovine kidney that bind to adenosine deaminase im... more We have isolated a complex of two proteins from bovine kidney that bind to adenosine deaminase immobilized on Sepharose 4B. One protein, with Mr = 110,000, comigrates on both PAGE and SDS-PAGE gels with complexing protein isolated from rabbit kidney by the method of Schrader et al. (Schrader, W.P., Harder, C. M., and Schrader, D. K. (1983) Comp. Biochem. Physiol. B Comp. Biochem. 75, 119-126). The second protein has a Mr = 70,000. Both proteins bind to the adenosine deaminase-Sepharose but not to a control resin of bovine serum albumin bound to Sepharose. Based on a comparison of partial and complete denaturation on SDS-PAGE the two proteins appear to be bound to each other. At adenosine concentrations of 0.5-1 mM the isolated complexing protein increases small subunit adenosine deaminase catalytic activity by 20-30%. There may be some inhibition of catalytic activity at low adenosine concentrations. We have designated the 110,000 Mr protein CP-I, the 70,000 Mr protein CP-II and the complex of these two CP.
Journal of Visualized Experiments, 2019
Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for d... more Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective timeper-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.
Biophysical Journal, 2018
Journal of Cell Science, 2018
Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF recep... more Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling, however they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interactions remain elusive. Number of molecules and Brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 by low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Si...
Progress in Biophysics and Molecular Biology
SPIE Proceedings, 1990
ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was exa... more ABSTRACT The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA's activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP's regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.
FEBS Letters, 1990
A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), I-Wetheno-[e... more A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), I-Wetheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (E-EHNA), is protonated at the active site of the enzyme. In E-EHNA [&=(4.06 + 1.00) 1O-6 M] part of the competive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with e-EHNA yielded the corrected excitation spectrum of o-EHNA at the active site of the enzyme. This spectrum mimics that of E-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site.
Drug Development Research, 2000
ABSTRACT While adenosine deaminase (ADA) is an established malignancy marker and its clinical inv... more ABSTRACT While adenosine deaminase (ADA) is an established malignancy marker and its clinical involvement in severe combined immunodeficiency (SCID) is understood, the biological significance of its binding to adenosine deaminase complexing protein (CP = DPPIV = CD26) remains enigmatic. The role of lipid–protein interactions in the modulation of ADA activity by membrane-bound CP was sought. ADA bound specifically to CP reconstituted in L-α-dimyristoylphosphatidylcholine (DMPC) or asolectin vesicles. Without CP, the binding and specific activity of ADA were negligible. In the presence of CP, the specific activity recovered to values similar to those in buffer. The addition of cholesterol increased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled vesicles and resulted in a “bell-shaped” dependence of the specific activity of ADA on cholesterol concentration. Vesicles with small cholesterol ratios mimicked Rouse sarcoma virus-transformed chick embryo fibroblasts (CEF) in that both had reduced ADA activity and increased membrane fluidity. In contrast to continuous Arrhenius plots of ADA activity in solution, free or bound to CP, ADA bound to CP reconstituted in DMPC vesicles exhibited two breaks, around 25 and 13°C, yielding three lines with similar apparent activation energies (Ea). Increased ADA activity of ∼30% was observed at each of these discontinuities. A model in which active site accessibility is dependent on membrane fluidity led to a successful simulation of the phase transitions. This model could also account for ADA reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts. Drug Dev. Res. 50:537–549, 2000. © 2000 Wiley-Liss, Inc.
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Papers by Valeria Caiolfa