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Kinesin cross-bridges between neurosecretory granules and microtubules in the mouse neurohypophysis
Neuroscience Letters 262 (1999) 69–71
Kinesin cross-bridges between neurosecretory granules and microtubules in the mouse neurohypophysis Takao Senda*, Wei Yu Department of Anatomy I, Nagoya University School of Medicine, Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan Received 4 January 1999; accepted 11 January 1999
Abstract Neurosecretory granules are conveyed along microtubules in the neurohypophysial axon. By both quick-freeze deep-etch and thin section electron microscopies, we found cross-bridges between the granules and microtubules. The length of the crossbridges (mean 26.6 ± 15.2 nm) was not uniform, and its histogram was multi-phasic showing the highest peak around 20 nm and several lower peaks in the range of 40–100 nm. This implies that cross-bridges are complexes of several kinds of constituents. Immunofluorescence microscopy showed the expression of a motor protein kinesin in the mouse neurohypophysis. Immunoelectron microscopy detected kinesin at the contact sites of neurosecretory granules to microtubules. These results suggest that kinesin is a major component of the cross-bridges, and involved in the neurosecretory granule transportation. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cross-bridge; Neurosecretory granule; Microtubule; Axonal transport; Kinesin; Neurohypophysis; Quick-freeze deepetch electron microscopy
Neurosecretory granules in the hypothalamo-neurohypophysial system are produced in the neuronal cell body located in the supraoptic and paraventricular nuclei, and transported through the axon toward the nerve terminal located in the neurohypophysis [1,6]. This transport is believed to be a part of the fast axonal transport, and dependent on the axonal microtubules. The axonal transport of membrane-bounded organelles has been studied intensively in the past 15 years, and thereby its molecular mechanisms are now successfully illustrated [3,4]. In neuronal axons, membrane organelles are linked to the microtubules by cross-bridges composed of kinesin or its family proteins that possess ATPase activity, and forced to translocate along the microtubules by the action of these motor proteins [3–5]. On the other hand, the precise mechanism of the axonal granule transportation in the hypothalamo-neurohypophysial system remains unknown. In the present study, we examined the mouse neurohypophysis by quick-freeze deep-etch and thin section electron microscopies to reveal * Corresponding author. Tel.: +81-52-7442001; fax.: +81-527442012; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)000 42-7
the relationship of the neurosecretory granules with the microtubules, and by immunoelectron microscopy to determine whether kinesin is involved in the transportation of the neurosecretory granules. Male ddy mice aged 8–10 weeks were used for this study. For quick-freeze deep-etch electron microscopy, fresh neurohypophyses resected from the decapitated mice were quickly-frozen with liquid helium as described previously [8], and stored in liquid nitrogen. The protocol of freezefracturing, deep-etching and preparation of the platinum replica was also described previously [8]. The replica of the mouse neurohypophysis was examined with an electron microscope (1200EX; JEOL, Tokyo, Japan). For conventional thin section electron microscopy, the mice anesthesized with sodium pentobarbital (30 mg/kg of body weight, i.p.) were perfused transcardially for 5 min with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The neurohypophysis was resected, and further immersed in the same fixative for 2 h followed by the post-fixation with 1% OsO4 for 1 h. Then, the specimens were dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections cut on an ultramicrotome (MT-1; Sorval,
1999 Elsevier Science Ireland Ltd. All rights reserved.
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T. Senda, W. Yu / Neuroscience Letters 262 (1999) 69–71
New Town, CT, USA) were stained with uranyl acetate and lead citrate, and examined with the electron microscope. For immunofluorescence microscopy, the mouse neurohypophysis was fixed with 4% paraformaldehyde, and its frozen sections were prepared as described previously [9]. The sections were pretreated with 1% bovine serum albumin (BSA) in PBS, and immunostained with monoclonal mouse anti-kinesin antibody (Sigma, St. Louis, MO, USA) diluted 1:500 with 1% BSA/PBS for 2 h. Control sections were incubated with non-immunized mouse serum in place of the antibody. The sections were labelled with FITC-conjugated goat anti-mouse IgG antibody (Sigma), and observed with a fluorescence microscope (Fluophoto; Nikon, Tokyo, Japan). For immunoelectron microscopy, the mouse neurohypophysis was fixed with the mixture of 4% paraformaldehyde and 0.1% glutaraldehyde, dehydrated in ethanol, and embedded in Lowicryl K4M resin as described previously [9]. The Lowicryl ultrathin sections were immunostained
with anti-kinesin antibody diluted 1:100 for 2 h, and then labelled with 5 nm-colloidal gold-conjugated goat antimouse IgG antibody (Amersham Japan, Tokyo, Japan). Control sections were incubated with non-immunized mouse serum in place of the antibody. The sections were stained with uranyl acetate and lead citrate, and examined with the electron microscope. Major cytoskeletal components in the neurohypophysial axon are microtubules and neurofilaments, 25–30 and 10– 15 nm in diameter, respectively, both of which were arranged almost in parallel with the axon [7]. In the mouse neurohypophysial axon, neurosecretory granules were present along and/or among the axonal cytoskeleton either solely, in line or in a cluster. We found by deep-etch electron microscopy that the neurosecretory granules were linked to the microtubules by cross-bridges (Fig. 1A). The cross-bridges were almost straight, and had no branching. The cross-bridges were also recognized in thin section electron microscopic images (Fig. 1B). We measured the length
Fig. 1. (A,B) Arrows indicate cross-bridges between neurosecretory granule (g) and microtubule (m) in the mouse neurohypophysis observed by quick-freeze deep-etch (A) and thin section (B) electron microscopies. (C) Expression of kinesin in the mouse neurohypophysis detected by immunofluorescence microscopy. (D) Arrows indicate the localization of kinesin (gold particles) at the contact site of a neurosecretory granule (g) with a microtubule (m) detected by immunoelectron microscopy. Scale bar, (A,B,D) 20 nm; (C) 20 mm.
T. Senda, W. Yu / Neuroscience Letters 262 (1999) 69–71
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cross-bridges are composed of motor proteins that make force to move the granules along microtubules. Actually, kinesin, a first-identified, microtubule-activated motor protein [2,10], was detected by immunoelectron microscopy at the contact sites between neurosecretory granules and microtubules, that is where the cross-bridges exist. Kinesin has been reported to form a cross-bridge as long as 25 nm between membrane organelles and microtubules [5]. In this study, the major peak of the cross-bridge length was around 20 nm. These results suggest that kinesin is a major component of neurohypophysial cross-bridges. The kinesin crossbridges may play important roles in the neurosecretory granule transportation through the neurohypophysial axon. We also found cross-bridges of more than 40 nm in length, and in the histogram of the length several lower peaks were seen between 40 and 100 nm. This indicates that the cross-bridges include other components apart from kinesin. In the past decade, a lot of motor proteins, referred to as kinesin-family proteins (KIFs), have been identified, and their roles in axonal transport have been elucidated [4]. They are also possible candidates for crossbridges though they were not identified in the present study.
Fig. 2. The size distribution of the cross-bridge length. The length of 141 cross-bridges was measured on thin section electron micrographs, and summarized in a histogram.
of the cross-bridges (141 in total) on thin section electron micrographs. The mean length of the cross-bridges was 26.6 ± 15.2 nm, and their length was not uniform. The histogram of the cross-bridge length showed a multi-phasic pattern; the highest peak was around 20 nm, and several lower peaks in the range of 40–100 nm (Fig. 2). These results from the morphometric analysis imply that crossbridges are complexes of several kinds of constituents. Kinesin is a motor protein involved in the microtubuledependent membrane organelle transport in both neuronal and non-neuronal cells [2–4,10]. To determine whether kinesin is involved in the neurosecretory granule transport, we next investigated expression and localization of kinesin in the neurohypophysis by both immunofluorescence and immunoelectron microscopic techniques. Kinesin was shown to be expressed in the mouse neurohypophysis (Fig. 1C). At the electron microscopic level, it was found that kinesin was associated with the neurosecretory granules, and often localized at the contact sites of the neurosecretory granules to the microtubules (Fig. 1D). No fluorescence and gold particle was seen in control sections of fluorescence and immunoelectron microscopies, respectively (data not shown). In the present study, we found cross-bridges between neurosecretory granules and microtubules by two kinds of electron microscopic techniques. It is postulated that the
This study was supported by the grants from the Ministry of Education, Science, Sports and Culture of Japan. [1] Bargmann, W., Neurosecretion, Int. Rev. Cytol., 19 (1966) 183–201. [2] Brady, S.T., A novel brain ATPase with properties expected for the fast axonal transport motor, Nature, 317 (1985) 73–75. [3] Hirokawa, N., Molecular architecture and dynamics of the neuronal cytoskeleton. In N. Hirokawa (Ed.), Neuronal Cytoskeleton, Wiley-Liss, New-York, 1991, 5–74. [4] Hirokawa, N., Kinesin and dynein superfamily proteins and the mechanism of organelle transport, Science, 279 (1998) 519– 526. [5] Hirokawa, N., Pfister, K.K., Yorifuji, H., Wagner, M.C., Brady, S.T. and Bloom, G.S., Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration, Cell, 56 (1989) 867–878. [6] Lederis, K., Neurosecretion and the functional structure of the neurohypophysis. In E. Knobil and W.H. Sawyer (Eds.), Handbook of Physiology, Section 7, Endocrinology, Vol. IV. The Pituitary Gland and its Neuroendocrine Control, Part 1, American Physiological Society, Washington DC, 1974, pp. 81–102. [7] Senda, T., Cytoskeletal organization in the neurohypophysial axons of rats, Med. Electron Microsc., 31 (1998) 94–99. [8] Senda, T. and Fujita, H., Ultrastructural aspects of quick-freezing deep-etching replica images of the cytoskeletal system in anterior pituitary secretory cells of rats and mice, Arch. Histol. Jpn., 50 (1987) 49–60. [9] Senda, T., Nishii, Y. and Fujita, H., Immunocytochemical localization of synapsin I in the adrenal medulla of rats, Histochemistry, 96 (1991) 25–30. [10] Vale, R.D., Reese, T.S. and Sheetz, M.P., Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility, Cell, 42 (1985) 39–50.
Kinesin cross-bridges between neurosecretory granules and microtubules in the mouse neurohypophysis Takao Senda*, Wei Yu Department of Anatomy I, Nagoya University School of Medicine, Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan Received 4 January 1999; accepted 11 January 1999
Abstract Neurosecretory granules are conveyed along microtubules in the neurohypophysial axon. By both quick-freeze deep-etch and thin section electron microscopies, we found cross-bridges between the granules and microtubules. The length of the crossbridges (mean 26.6 ± 15.2 nm) was not uniform, and its histogram was multi-phasic showing the highest peak around 20 nm and several lower peaks in the range of 40–100 nm. This implies that cross-bridges are complexes of several kinds of constituents. Immunofluorescence microscopy showed the expression of a motor protein kinesin in the mouse neurohypophysis. Immunoelectron microscopy detected kinesin at the contact sites of neurosecretory granules to microtubules. These results suggest that kinesin is a major component of the cross-bridges, and involved in the neurosecretory granule transportation. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cross-bridge; Neurosecretory granule; Microtubule; Axonal transport; Kinesin; Neurohypophysis; Quick-freeze deepetch electron microscopy
Neurosecretory granules in the hypothalamo-neurohypophysial system are produced in the neuronal cell body located in the supraoptic and paraventricular nuclei, and transported through the axon toward the nerve terminal located in the neurohypophysis [1,6]. This transport is believed to be a part of the fast axonal transport, and dependent on the axonal microtubules. The axonal transport of membrane-bounded organelles has been studied intensively in the past 15 years, and thereby its molecular mechanisms are now successfully illustrated [3,4]. In neuronal axons, membrane organelles are linked to the microtubules by cross-bridges composed of kinesin or its family proteins that possess ATPase activity, and forced to translocate along the microtubules by the action of these motor proteins [3–5]. On the other hand, the precise mechanism of the axonal granule transportation in the hypothalamo-neurohypophysial system remains unknown. In the present study, we examined the mouse neurohypophysis by quick-freeze deep-etch and thin section electron microscopies to reveal * Corresponding author. Tel.: +81-52-7442001; fax.: +81-527442012; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)000 42-7
the relationship of the neurosecretory granules with the microtubules, and by immunoelectron microscopy to determine whether kinesin is involved in the transportation of the neurosecretory granules. Male ddy mice aged 8–10 weeks were used for this study. For quick-freeze deep-etch electron microscopy, fresh neurohypophyses resected from the decapitated mice were quickly-frozen with liquid helium as described previously [8], and stored in liquid nitrogen. The protocol of freezefracturing, deep-etching and preparation of the platinum replica was also described previously [8]. The replica of the mouse neurohypophysis was examined with an electron microscope (1200EX; JEOL, Tokyo, Japan). For conventional thin section electron microscopy, the mice anesthesized with sodium pentobarbital (30 mg/kg of body weight, i.p.) were perfused transcardially for 5 min with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The neurohypophysis was resected, and further immersed in the same fixative for 2 h followed by the post-fixation with 1% OsO4 for 1 h. Then, the specimens were dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections cut on an ultramicrotome (MT-1; Sorval,
1999 Elsevier Science Ireland Ltd. All rights reserved.
70
T. Senda, W. Yu / Neuroscience Letters 262 (1999) 69–71
New Town, CT, USA) were stained with uranyl acetate and lead citrate, and examined with the electron microscope. For immunofluorescence microscopy, the mouse neurohypophysis was fixed with 4% paraformaldehyde, and its frozen sections were prepared as described previously [9]. The sections were pretreated with 1% bovine serum albumin (BSA) in PBS, and immunostained with monoclonal mouse anti-kinesin antibody (Sigma, St. Louis, MO, USA) diluted 1:500 with 1% BSA/PBS for 2 h. Control sections were incubated with non-immunized mouse serum in place of the antibody. The sections were labelled with FITC-conjugated goat anti-mouse IgG antibody (Sigma), and observed with a fluorescence microscope (Fluophoto; Nikon, Tokyo, Japan). For immunoelectron microscopy, the mouse neurohypophysis was fixed with the mixture of 4% paraformaldehyde and 0.1% glutaraldehyde, dehydrated in ethanol, and embedded in Lowicryl K4M resin as described previously [9]. The Lowicryl ultrathin sections were immunostained
with anti-kinesin antibody diluted 1:100 for 2 h, and then labelled with 5 nm-colloidal gold-conjugated goat antimouse IgG antibody (Amersham Japan, Tokyo, Japan). Control sections were incubated with non-immunized mouse serum in place of the antibody. The sections were stained with uranyl acetate and lead citrate, and examined with the electron microscope. Major cytoskeletal components in the neurohypophysial axon are microtubules and neurofilaments, 25–30 and 10– 15 nm in diameter, respectively, both of which were arranged almost in parallel with the axon [7]. In the mouse neurohypophysial axon, neurosecretory granules were present along and/or among the axonal cytoskeleton either solely, in line or in a cluster. We found by deep-etch electron microscopy that the neurosecretory granules were linked to the microtubules by cross-bridges (Fig. 1A). The cross-bridges were almost straight, and had no branching. The cross-bridges were also recognized in thin section electron microscopic images (Fig. 1B). We measured the length
Fig. 1. (A,B) Arrows indicate cross-bridges between neurosecretory granule (g) and microtubule (m) in the mouse neurohypophysis observed by quick-freeze deep-etch (A) and thin section (B) electron microscopies. (C) Expression of kinesin in the mouse neurohypophysis detected by immunofluorescence microscopy. (D) Arrows indicate the localization of kinesin (gold particles) at the contact site of a neurosecretory granule (g) with a microtubule (m) detected by immunoelectron microscopy. Scale bar, (A,B,D) 20 nm; (C) 20 mm.
T. Senda, W. Yu / Neuroscience Letters 262 (1999) 69–71
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cross-bridges are composed of motor proteins that make force to move the granules along microtubules. Actually, kinesin, a first-identified, microtubule-activated motor protein [2,10], was detected by immunoelectron microscopy at the contact sites between neurosecretory granules and microtubules, that is where the cross-bridges exist. Kinesin has been reported to form a cross-bridge as long as 25 nm between membrane organelles and microtubules [5]. In this study, the major peak of the cross-bridge length was around 20 nm. These results suggest that kinesin is a major component of neurohypophysial cross-bridges. The kinesin crossbridges may play important roles in the neurosecretory granule transportation through the neurohypophysial axon. We also found cross-bridges of more than 40 nm in length, and in the histogram of the length several lower peaks were seen between 40 and 100 nm. This indicates that the cross-bridges include other components apart from kinesin. In the past decade, a lot of motor proteins, referred to as kinesin-family proteins (KIFs), have been identified, and their roles in axonal transport have been elucidated [4]. They are also possible candidates for crossbridges though they were not identified in the present study.
Fig. 2. The size distribution of the cross-bridge length. The length of 141 cross-bridges was measured on thin section electron micrographs, and summarized in a histogram.
of the cross-bridges (141 in total) on thin section electron micrographs. The mean length of the cross-bridges was 26.6 ± 15.2 nm, and their length was not uniform. The histogram of the cross-bridge length showed a multi-phasic pattern; the highest peak was around 20 nm, and several lower peaks in the range of 40–100 nm (Fig. 2). These results from the morphometric analysis imply that crossbridges are complexes of several kinds of constituents. Kinesin is a motor protein involved in the microtubuledependent membrane organelle transport in both neuronal and non-neuronal cells [2–4,10]. To determine whether kinesin is involved in the neurosecretory granule transport, we next investigated expression and localization of kinesin in the neurohypophysis by both immunofluorescence and immunoelectron microscopic techniques. Kinesin was shown to be expressed in the mouse neurohypophysis (Fig. 1C). At the electron microscopic level, it was found that kinesin was associated with the neurosecretory granules, and often localized at the contact sites of the neurosecretory granules to the microtubules (Fig. 1D). No fluorescence and gold particle was seen in control sections of fluorescence and immunoelectron microscopies, respectively (data not shown). In the present study, we found cross-bridges between neurosecretory granules and microtubules by two kinds of electron microscopic techniques. It is postulated that the
This study was supported by the grants from the Ministry of Education, Science, Sports and Culture of Japan. [1] Bargmann, W., Neurosecretion, Int. Rev. Cytol., 19 (1966) 183–201. [2] Brady, S.T., A novel brain ATPase with properties expected for the fast axonal transport motor, Nature, 317 (1985) 73–75. [3] Hirokawa, N., Molecular architecture and dynamics of the neuronal cytoskeleton. In N. Hirokawa (Ed.), Neuronal Cytoskeleton, Wiley-Liss, New-York, 1991, 5–74. [4] Hirokawa, N., Kinesin and dynein superfamily proteins and the mechanism of organelle transport, Science, 279 (1998) 519– 526. [5] Hirokawa, N., Pfister, K.K., Yorifuji, H., Wagner, M.C., Brady, S.T. and Bloom, G.S., Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration, Cell, 56 (1989) 867–878. [6] Lederis, K., Neurosecretion and the functional structure of the neurohypophysis. In E. Knobil and W.H. Sawyer (Eds.), Handbook of Physiology, Section 7, Endocrinology, Vol. IV. The Pituitary Gland and its Neuroendocrine Control, Part 1, American Physiological Society, Washington DC, 1974, pp. 81–102. [7] Senda, T., Cytoskeletal organization in the neurohypophysial axons of rats, Med. Electron Microsc., 31 (1998) 94–99. [8] Senda, T. and Fujita, H., Ultrastructural aspects of quick-freezing deep-etching replica images of the cytoskeletal system in anterior pituitary secretory cells of rats and mice, Arch. Histol. Jpn., 50 (1987) 49–60. [9] Senda, T., Nishii, Y. and Fujita, H., Immunocytochemical localization of synapsin I in the adrenal medulla of rats, Histochemistry, 96 (1991) 25–30. [10] Vale, R.D., Reese, T.S. and Sheetz, M.P., Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility, Cell, 42 (1985) 39–50.