Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.

Background:The functional importance of the C2 insert-containing isoform of nonmuscle myosin II-C is not known. Results: During the neuritogenesis of Neuro-2a cells, NM II-C1C2 becomes the predominant isoform of NM II-C. Decreasing NM II-C1C2 expression leads to shortening of neurites. Conclusion: NM II-C1C2 plays a role in the later stage of neuritogenesis. Significance: This work contributes to an understanding of the functional importance of NM II-C1C2.

BackgroundFor a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.MethodsGAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.ResultHere, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.ConclusionPKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material, which is available to authorized users.

One molecular cue that regulates cellular protrusions such as blebbing and lamellipodia in tumor cells has been less explored than other environmental factors. NM II-A induces blebbing and NM II-C1 induces lamellipodia in tumor cells. NM-II isoforms can change the protrusive activity of a tumor cell.

The septation initiation network (SIN), comprising a GTPase and a cascade of three protein kinases, regulates cell division in fission yeast , but questions remain about its influence on cytokinesis. Here, we made quantitative measurements of the numbers of Cdc7p kinase molecules (a marker for SIN activity) on spindle pole bodies (SPBs), and on the timing of assembly, maturation and constriction of contractile rings via six different proteins tagged with fluorescent proteins. When SIN activity is low in mutant cells at 32°C, cytokinetic nodes formed contractile rings ∼3 min slower than wild-type cells. During the maturation period, these rings maintained normal levels of the myosin-II mEGFP-Myo2p but accumulated less of the F-BAR protein Cdc15p-GFP than in wild-type cells. The Cdc15p-GFP fluorescence then disintegrated into spots as mEGFP-Myo2p dissociated slowly. Some rings started to constrict at the normal time, but most failed to complete constriction. When high SIN activity persists far longer than normal on both SPBs in mutant cells at 32°C, contractile rings assembled and constricted normally, but disassembled slowly, delaying cell separation.