Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule "force-velocity curves" have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report forcevelocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37°C, three parameters were measured-velocity (v), radius (a), and effective cytoplasmic viscosity (η′)-as they applied to moving vesicles. These parameters were combined in Stokes' equation, F = 6πaη′v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force-velocity curve for a single motor agrees with previously reported in vitro single motor force-velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.
Determining in vivo force-velocity relationships of motor proteins is a critical step toward clarifying how they accomplish intracellular transport. We show that in vivo force-velocity curves corresponding to an estimated 1, 2, and 3 motors-per-vesicle can be constructed by tracking and sizing transported vesicles. The force range for these curves would normally be constrained by diffraction limited diameter measurements. However, we present a new method that uses the image intensity obtained with differential interference contrast microscopy as a proxy for vesicle diameters smaller than the diffraction limit. We calibrate this novel sizing method in vitro with polystyrene microsphere standards and apply it in vivo to vesicles. The resulting diameter vs. velocity data for large, small, and sub-diffraction limited vesicles is used to construct force-velocity curves that extend the force range of our previous curves. These extended 1-, 2-, and 3-motor in vivo curves qualitatively agree with a simple model of load sharing for motors that jointly transport a single vesicle.
The standard codon table is a primary tool for basic understanding of molecular biology. In the minds of many, the table's orderly arrangement of bases and amino acids is synonymous with the true genetic code, i.e., the biological coding principle itself. However, developments in the field reveal a much more complex and interesting picture. In this article, we review the traditional codon table and its limitations in light of the true complexity of the genetic code. We suggest the codon table be brought up to date and, as a step, we present a novel superposition of the BLOSUM62 matrix and an allowed point mutation matrix. This superposition depicts an important aspect of the true genetic code-its ability to tolerate mutations and mistranslations.
Primary neuron cultures are widely used in research due to the ease and usefulness of observing individual cells. Therefore, it is vital to understand how variations in culture conditions may affect neuron physiology. One potential variation for cultured neurons is a change in intracellular transport. As transport is necessary for the normal delivery of organelles, proteins, nucleic acids, and lipids, it is a logical indicator of a cell's physiology. We test the hypothesis that organelle transport may change with varying in vitro population densities, thus indicating a change in cellular physiology. Using a novel background subtraction imaging method we show that, at 5 days in vitro (DIV), transport of vesicular organelles in embryonic rat spinal cord neurons is positively correlated with cell density. When density increased 6.5 fold, the number of transported organelles increased 2.2 ± 0.3 fold. Intriguingly, this effect was not observable at 3-4 DIV. These results show a significant change in cellular physiology with a relatively small change in plating procedure; this indicates that cells appearing to be morphologically similar, and at the same DIV, may still suffer from a great degree of variability.
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