Amato P A, Loizzi R F
Cell Motil. 1981;1(3):329-47. doi: 10.1002/cm.970010305.
Cytochalasin B, a microfilament-altering drug, inhibits lactose synthesis in lactating guinea pig mammary gland [Biochim. Biophys. Acta 392:20, 1975] but not primary by inhibiting glucose transport [Eur. J. Cell Biol. 20:150, 1979]. In order to study the possible role of microfilaments in lactose synthesis and secretion, we isolated both the alveolar (milk-secreting) and myoepithelial (contractile) cells from lactating mammary gland. Light microscopy shows that the alveolar cell fraction (viability approximately 71%) is homogenous and that the cells retain strong polarity of secretory structures in the apical region. Two proteins were extracted from the alveolar cell fraction. One (mol wt 42,000) comigrates with skeletal muscle actin on SDS-PAGE gels. The other, a high-molecular-weight (180,000) protein (HMWP) may be analogous to actin-binding protein or clathrin. An extract from the myoepithelial cell fraction also contains a protein that comigrates with actin but no HMWP. Whole tissue extract contains the 42K protein, and a 185K HMWP. Examination of the alveolar cell extract by electron microscopic (EM) negative staining revealed meshworks of multistranded, interconnecting filaments, with attached globular structures (100-200 A) (possibly the HMWP) and single filaments (40-60 A diameter) branching off. To localize these filamentous structures in situ, whole tissue was glycerinated and incubated with rabbit skeletal muscle heavy meromyosin (HMM). Masses of filaments in myoepithelial cells served as convenient standards for HMM decoration. Decorated filaments have cross-arms or projections, unlike the narrow, smooth filaments of control tissue. Decorated filaments in alveolar cells are located beneath the plasma membrane, in close association with secretory vacuoles, and near the Golgi apparatus; filaments near the latter two are often oriented perpendicular to the plasma membrane. Microvesicles are embedded in meshworks under the plasmalemma and near the Golgi apparatus. Intermediate-sized (85-115 A diameter), non-decorated filaments diverge from the meshworks of decorated filaments. Micro-vesicles are associated with intermediate-sized filaments as well. The association of actin-like filaments with secretory vacuoles and microvesicles and their location in areas of the cell concerned with biosynthetic activities suggest a possible function in the intracellular transport of secretory products.
细胞松弛素B是一种能改变微丝的药物，它可抑制泌乳豚鼠乳腺中的乳糖合成[《生物化学与生物物理学学报》392:20，1975]，但并非主要通过抑制葡萄糖转运来实现[《欧洲细胞生物学杂志》20:150，1979]。为了研究微丝在乳糖合成和分泌中可能发挥的作用，我们从泌乳乳腺中分离出了腺泡（分泌乳汁的）细胞和肌上皮（收缩性的）细胞。光学显微镜显示，腺泡细胞组分（活力约为71%）是均匀的，并且细胞在顶端区域保留了分泌结构的强极性。从腺泡细胞组分中提取了两种蛋白质。一种（分子量42,000）在SDS - PAGE凝胶上与骨骼肌肌动蛋白迁移率相同。另一种是高分子量（180,000）的蛋白质（HMWP），可能类似于肌动蛋白结合蛋白或网格蛋白。肌上皮细胞组分的提取物中也含有一种与肌动蛋白迁移率相同的蛋白质，但不含HMWP。全组织提取物含有42K蛋白质和一种185K的HMWP。通过电子显微镜（EM）负染色对腺泡细胞提取物进行检查，发现有多股相互连接的细丝组成的网络，上面附着有球状结构（100 - 200 Å）（可能是HMWP）以及从其分支出来的单丝（直径40 - 60 Å）。为了在原位定位这些丝状结构，将全组织用甘油处理并与兔骨骼肌重酶解肌球蛋白（HMM）一起孵育。肌上皮细胞中的大量细丝作为HMM标记的便利标准。被标记的细丝有横臂或突起，这与对照组织中狭窄、光滑的细丝不同。腺泡细胞中的被标记细丝位于质膜下方，与分泌泡紧密相连，并靠近高尔基体；后两者附近的细丝通常与质膜垂直排列。微泡嵌入质膜下和高尔基体附近的网络中。中等大小（直径85 - 115 Å）、未被标记的细丝从被标记细丝的网络中分支出来。微泡也与中等大小的细丝相关联。类似肌动蛋白的细丝与分泌泡和微泡的关联以及它们在细胞中与生物合成活动相关区域的位置表明，它们在分泌产物的细胞内运输中可能具有作用。
