Date of Award
1993
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Circulating insect hemocytes become adherent to foreign surfaces and to one another during immune and wound healing responses. Hemocytes form various types of intercellular junctions, including gap junctions, as they encapsulate large foreign objects in the hemocoel. This study focuses on two aspects of hemocyte behaviour in vitro: hemocyte/substrate adhesion and gap junction formation.;Cockroach (Periplaneta americana) hemocytes resuspendad in medium containing purified lipophorin, either in the presence or absence of Ca{dollar}\sp{lcub}2+{rcub},{dollar} remained non-adherent to glass coverslips and retained a discoid morphology in vitro for at least 30 minutes. In contrast, hemocytes incubated either with or without Ca{dollar}\sp{lcub}2+{rcub}{dollar} for 30 minutes in lipophorin-deficient plasma, BSA, or saline alone adhered and flattened onto coverslips. The finding that lipophorin stabilized hemocytes in vitro is important since to date it has been difficult to maintain hemocytes in a non-adhesive state in vitro.;Calf serum inhibited hemocyte adhesion in the presence and absence of Ca{dollar}\sp{lcub}2+{rcub}.{dollar} Hemocyte adhesion was inhibited by the human plasma lipoprotein apoB-100, but not by apoA-I, apoA-II, apoC-I, apoC-II, apoE, or mouse IgG. Of eight synthetic peptides with sequences corresponding to short regions (15 to 43 amino acids) of human apoB-100, one peptide corresponding to amino acids 4154-4189 inhibited hemocyte adhesion. Human apoB-89, a truncated form of apoB-100 lacking the region containing 4154-4189, was also active, indicating that one or more additional sites exist on apoB-89 which are involved in inhibiting hemocyte adhesion.;Freeze-fracture replicas of hemocyte aggregates fixed 5 minutes after bleeding were seen to contain E-face particles coalescing to form gap junctional plaques. Dye passage was detected between carboxyfluorescein diacetate-labelled and unlabelled hemocytes within 3 minutes of bleeding, when the cells made contact as they flattened rapidly onto coverslips. Dye-coupling was detected in the absence of Ca{dollar}\sp{lcub}2+{rcub},{dollar} indicating that involvement of Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent cell adhesion molecules is not a prerequisite of gap junction formation in hemocytes. Hemocytes from distantly related insects (cockroach and moth) formed functional gap junctions with each other, suggesting sequence homology among gap junction proteins in insects. Using a quantitative dye-transfer assay, it was determined that trypsin reduced the formation of functional gap junctions between hemocytes, but only if the surfaces of both 'dye-donor' and 'dye-recipient' cells were trypsinized. This could be due to cleaving of GJ protein or cell adhesion molecules on the hemocyte surface.;Hemocyte flattening in vitro was delayed by plating cells on lipophorin-coated coverslips in order to facilitate patch-clamp studies of these cells. When double whole-cell voltage-clamp was used to measure gap junction formation between cells which were pushed together, electrical coupling was detected within one second of cell-cell contact. Junctional conductance increased in staircase fashion with steps corresponding to single channel conductances of 345 pS.
Recommended Citation
Coodin, Sheppy Israel, "Substrate Adhesion And Gap Junction Formation By Insect Hemocytes In Vitro" (1993). Digitized Theses. 2267.
https://ir.lib.uwo.ca/digitizedtheses/2267