| The diatom Achnanthes longipes attaches to surfaces by secreting extracellular matrix (ECM) during motility and via more permanent extracellular adhesive structures termed stalks. The stalks of the diatom consist of a cell-associated collar, a surface adhered pad, and a shaft, which separates the pad from the cell.; An integrated approach including electron microscopy cryo-techniques, video microscopy and immunocytochemistry were utilized to investigate biosynthesis and stalk polymer distribution and organization in the native state. Time-lapse video microscopy revealed stalk polymer secretion occurred at an undifferentiated extrusion zone associated with the raphid valve at the cell pole. This extrusion zone (EZ) was characterized by active cytoplasmic activities such as discharge of adhesive encapsulated in vesicles at the PM, and aggregation of granular inclusions near the raphe terminus. High-pressure freezing/freeze substitution (HPF/FS) provided good preservation and revealed cytoplasmic vesicles involved in deposition of extracellular polymers. A continuous, multi-layered diatotepic layer surrounding the protoplasm and internal to the frustule was visualized with freeze-fracture/cryo-field emission SEM and by TEM of HPF/FS preserved material. Extracellular polymers deposited at the plasma membrane passed through this layer and the frustule during motility and stalk assembly. During formation of a mature and distinct structured stalk, excreted polymers are self-assembled and oriented within an assembly zone, an area external to the frustule and within the collar region. Monoclonal antibodies that selectively recognized various epitopes on the proteoglycans which make up the stalk were coupled with electron dense probes and applied to analyze stalk polymer processing, secretion and extracellular assembly. Immuno-gold labeling showed both low-sulfated and high-sulfated extracellular polymers synthesized and stored in vesicles associated with the Golgi apparatus before being secreted. Chemically unique regions of the complex, multi-layered stalk were revealed by antibody labeling. Immunocytochemistry results demonstrated that high-sulfate ECM polymers are extruded through the raphe and assembled into the central core, whereas low-sulfate polymers are extruded through the frustule pores near the raphe terminus, forming the outer layers of the stalk. Microscopy demonstrated that the multi-laminate stalk is made of microfibrillar and amorphous non-fibrillar material. The central core is mainly composed of microfibrils oriented perpendicularly to the length of the stalk reflecting the stalk is flexible and resistance to desiccation maybe contributed to this region. The outer layers of the stalk are composed of randomly oriented microfibrils and condensed and tightly knit fibril bundles oriented parallel to the length of the stalk, which add strength to flexible stalk structures. |
