Manu Tewari

Human skeletal myosin heavy chain genes are tightly linked in the order embryonic-IIa-IId/x-IIb-perinatal-extraocular

Myosin heavy chain (MyHC) is the major contractile protein of muscle. We report the first complete cosmid cloning and definitive physical map of the tandemly linked human skeletal MyHC genes at 17p13.1. The map provides new information on the order, size, and relative spacing of the genes, and it resolves uncertainties about the two fastest twitch isoforms. The physical order of the genes is demonstrated to contrast with the temporal order of their developmental expression. Furthermore, nucleotide sequence comparisons allow an approximation of the relative timing of five ancestral duplications that created distinct genes for the six isoforms. A firm foundation is provided for molecular analysis in patients with suspected primary skeletal myosinopathies and for detailed modelling of the hypervariable surface loops which dictate myosins kinetic properties.

The myosin filament XV assembly: contributions of 195 residue segments of the myosin rod and the eight C-terminal residues

SummaryA mixture of two peptides of approximately Mr 13 000 has been isolated from a papain digest of LC2 deficient myosin. The peptides assemble into highly ordered aggregates which in one view are made up of strands of pairs of dots with an average side to side spacing of 13.0 nm and an average axial repeat of 9.0 nm. In another view there are strands of single dots with a side-to-side spacing of 7.8 nm and an axial repeat of 9.1 nm. From N-terminal peptide sequencing, the two peptides have been shown to come from regions of the myosin rod displaced by 195 residues. We have shown that either peptide alone can assemble to form the same aggregates. The 195 residue displacement of the Mr 13 000 peptides corresponds closely to the 196 residue repeat of charges along the myosin rod. This finding permits us to designate 195 residue segments of the myosin rod and to relate assembly characteristics directly to the similar 195 residue segments and 196 residue charge repeat. The most C-terminal 195 residue segment carries information for assembly into helical strands. The contiguous 195 residue segment, in major part, carries information for the unipolar assembly, characteristic of the assembly in each half of the myosin filament. The next contiguous 195 residue segment, in major part, carries information for bipolar assembly which is characteristic of the bare zone region of the filament; and for the transition from the bipolar bare zone to unipolar assembly. The effect of the eight C-terminal residues of the myosin rod on the assembly of the contiguous 195 residues has also been studied. The entire fragment of 195 + eight C-terminal residues assembled to form helical strands with an axial repeat of 30 nm. Successive deletion of charged residues changed the axial repeat of the helical strands suggesting that the charged residues at the C-terminus are involved in determining the pitch in the helical assembly of the contiguous 195 residues.

Chapter 9:Anti-Cancer Polymersomes

Nature has evolved many solutions to many problems of transport at the cellular and sub-cellular scale. Small lipid vesicles or liposomes bud from cell membranes and help traffic a wide range of biomolecules both within cells and outside cells. Fluidity, flexibility, and dynamics of these carriers l...

Progerin phosphorylation in interphase is lower and less mechanosensitive than lamin-A,C in iPS-derived mesenchymal stem cells

ABSTRACT Interphase phosphorylation of lamin-A,C depends dynamically on a cells microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for diseased forms such as progerin, a permanently farnesylated mutant of LMNA that accelerates aging of stiff and mechanically stressed tissues. Here, fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs). The stoichiometry of total A-type lamins (including progerin) versus B-type lamins measured for Progeria iPS-MSCs prove similar to that of normal MSCs, with total A-type lamins more abundant than B-type lamins. However, progerin behaves more like farnesylated B-type lamins in mechanically-induced segregation from nuclear blebs. Phosphorylation of progerin at multiple sites in iPS-MSCs cultured on rigid plastic is also lower than that of normal lamin-A and C. Reduction of nuclear tension upon i) cell rounding/detachment from plastic, ii) culture on soft gels, and iii) inhibition of actomyosin stress increases phosphorylation and degradation of lamin-C > lamin-A > progerin. Such mechano-sensitivity diminishes, however, with passage as progerin and DNA damage accumulate. Lastly, transcription-regulating retinoids exert equal effects on both diseased and normal A-type lamins, suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.

DNA damage in 3D constricted migration or after lamin-A depletion in 2D: shared mechanisms of repair factor mis-localization under nuclear stress

Cells that migrate through small, rigid pores and that have normal levels of the nuclear structure protein lamin-A exhibit an increase in DNA damage, which is also observed with lamin-A depletion in diseases such as cancer and with many lamin-A mutations. Here we show nuclear envelope rupture is a shared feature that increases in standard culture after lamin-A knockdown, which causes nuclear loss of multiple DNA repair factors and increased DNA damage. Some repair factors are merely mis-localized to cytoplasm whereas others are partially depleted unless rescued by lamin-A expression. Compared to standard cultures on rigid glass coverslips, the growth of lamin-A low cells on soft matrix relaxes cytoskeletal stress on the nucleus, suppresses the mis-localization of DNA repair factors, and minimizes DNA damage nearly to wildtype levels. Conversely, constricted migration of the lamin-A low cells causes abnormally high levels of DNA damage, consistent with sustained loss of repair factors. The findings add insight into why monogenic progeroid syndromes that often associate with increased DNA damage and predominantly impact cells in stiff tissues result from mutations only in lamin-A or DNA repair factors.

511. Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth, immunosuppression, and are nonphagocytic. In our studies, less differentiated donor marrow phagocytes are engineered to target tumors and selectively phagocytose cancer cells. Xenograft tumors were made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface ‘self’ markers that limit the phagocytosis of these cells, we inhibited the ‘self’ receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of ‘self’-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >85% of macrophages that were ‘self’-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~30-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of ‘self’ inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of ‘self’ signaling.