Immunofluorescent Localization of Ca²⁺-Sensor Proteins in The Somatic Motor Muscles of The Earthworm Lumbricus terrestris

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Abstract

The method of immunofluorescent staining of earthworm somatic muscle samples showed the presence of calmodulin, Ca²⁺-calmodulin-dependent protein kinases type 1 and type 2, synaptotagmin type 2 and type 7 and calcineurin A. These proteins are detected in both synaptic and extrasynaptic regions of the motor muscle. However, for synaptotagmin type 2 and type 7, calcineurin A, their predominant localization in the area of neuromuscular synapses has been established. Besides, synaptic localization for synaptotagmin 7 and calcineurin A is most clearly expressed.

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About the authors

L. F. Nurullin

Kazan Institute of Biochemistry and Biophysics, Federal Research Center “Kazan Scientific Center of Russian Academy of Sciences”; Kazan State Medical University

Author for correspondence.
Email: lenizn@yandex.ru
Russian Federation, Kazan; Kazan

N. D. Almazov

Kazan State Medical University

Email: lenizn@yandex.ru
Russian Federation, Kazan

E. M. Volkov

Kazan State Medical University

Email: euroworm@mail.ru
Russian Federation, Kazan

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2. Fig. 1. Fluorescent triple labeling of a preparation of somatic muscle cells of the earthworm Lumbricus terrestris. (a) — Immunolabeling with antibodies to calmodulin (green); arrows indicate areas of more intense labeling; (b) — immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) — labeling of TMP-α-B of postsynaptic nAChRs (blue); (d) — superposition of images (a) and (b); (e) — superposition of images (a) and (c); (f) — superposition of images (a), (b), and (c). Scale bar 20 µm.

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3. Fig. 2. Detection of Ca²⁺-calmodulin-dependent protein kinase type 1 by fluorescent triple labeling of earthworm somatic muscle cell preparation. (a) — Immunolabeling with antibodies to Ca²⁺-calmodulin-dependent protein kinase type 1 (green); arrows indicate sites of more intense labeling; (b) — immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) — TMP-α-B labeling of postsynaptic nAChRs (blue); (d) — superposition of images (a) and (b); (e) — superposition of images (a) and (c); (f) — superposition of images (a), (b) and (c). Scale bar 20 µm.

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4. Fig. 3. The presence of Ca²⁺-calmodulin-dependent protein kinase type 2 in triple fluorescent labeling of earthworm somatic muscle cell preparation. (a) – Immunolabeling with antibodies to Ca²⁺-calmodulin-dependent protein kinase type 2 (green); (b) – immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) – labeling of nAChRs using TMP-α-B (blue); (d) – superposition of images (a) and (b); (e) – superposition of images (a) and (c); (f) – superposition of images (a), (b) and (c). Scale bar 20 µm.

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5. Fig. 4. Detection of synaptotagmin type 2 by fluorescent triple labeling of earthworm somatic muscle cell preparation. (a) Immunolabeling with antibodies to synaptotagmin 2 (green); (b) Immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) Labeling of nAChRs with TMP-α-B (blue); (d) Overlay of images (a) and (b); (e) Overlay of images (a) and (c); (f) Overlay of images (a), (b), and (c). Scale bar 20 µm.

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6. Fig. 5. Detection of synaptotagmin type 7 by fluorescent triple labeling of earthworm somatic muscle cell preparation. (a) – Immunolabeling with antibodies to synaptotagmin 7 (green); arrows indicate areas of more intense staining; (b) – immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) – labeling of nAChRs using TMP-α-B (blue); (d) – superposition of images (a) and (b); (e) – superposition of images (a) and (c); (f) – superposition of images (a), (b) and (c). Scale bar 20 µm.

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7. Fig. 6. The presence of calcineurin A in fluorescent triple labeling of earthworm somatic muscle cell preparation. (a) – Immunolabeling with antibodies to calcineurin A (green); (b) – immunolabeling with antibodies to the presynaptic protein synaptophysin (red); (c) – labeling of nAChRs using TMP-α-B (blue); (d) – superposition of images (a) and (b); (e) – superposition of images (a) and (c); (f) – superposition of images (a), (b) and (c). Scale bar 20 µm.

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