Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes

Abstract

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function

“…In intact hepatocytes exposed to PP inhibitors, we have shown that MT-dependent vesicle movements are significantly impeded [9]. These changes are not associated with any common changes in the MT array ( Figure 3) [9].…”

“…Previous work has shown that 500 nM MCY or 250 nM OKA results in comparable reductions in MT-based vesicle movements and receptor-mediated endocytosis [9,10]. As shown in Figure 1, the phosphorylation of several components of the cytoplasmic Figure 1 Cytoplasmic dynein-associated polypeptides phosphorylated in hepatocytes exposed to OKA or MCY Hepatocytes were labelled with 32 P[P i ] (0.3 mCi /1.5i10 6 cells, 2 h) prior to treatment with PP inhibitors (250 nM OKA or 500 nM MCY, 60 min), and lysates were exposed to UV light in the absence (no UV-VAN) or presence (UV-VAN) of 100 mM VAN and 2 mM MgATP.…”

“…Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN, U.S.A.) fed ad lib., weighing 240-300 g body weight, were used to isolate rat hepatocytes aseptically by the method of Moldeus et al [15] and as described previously [9,10]. Hepatocytes were cultured 24 h before use.…”

“…Rat hepatocytes seeded on to collagen-coated glass coverslips were exposed to the treatments outlined above, fixed and processed as described previously [9] using a mouse monoclonal anti-(α-tubulin) antibody (YOL1\34) and an FITC-conjugated goat anti-mouse secondary antibody.…”

“…In hepatocytes, we have previously shown that even a modest degree of PP inhibition by low doses of microcystin (MCY) or okadaic acid (OKA) (specific inhibitors of PP1, PP2A, and also PP4 and PP5 [8]) results in significantly impaired movement of vesicles along MTs [9], events which are correlated with major reductions in receptor-mediated endocytosis of transferrin [10]. At doses where MT-dependent vesicle movements are impaired, there are no major changes in MT integrity ; higher doses and\or longer times of exposure to MCY or OKA are needed to evoke the well-described disruption of the cytoskeleton by these inhibitors [11].…”