Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner.

DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

J Cell Biol 1998 Feb 23;140(4):779-93
A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells.
Yoon Y, Pitts KR, Dahan S, McNiven MA.

Identification and cellular localization
Dynamins are 100 kD GTPases that participate in the formation of nascent vesicles. Three forms of dynamin have been identified in mammalian tissues thus far while recent studies in yeast have demonstrated that the dynamin-related proteins Vps1p and Dnm1p participate in protein sorting at the Golgi and late endosomal compartments, respectively.

Using a purified polyclonal peptide antibody to a conserved domain of dynamin, we identified and isolated an immunoreactive 80 kD protein from rat liver homogenates. Amino acid sequences of tryptic peptides from this protein were determined and showed striking similarity to dynamin. Further screening of a rat cDNA library identified a single open reading frame encoding 755 amino acids with a calculated molecular weight of 84 kD. This protein shares 37% and 36% homology with rat dynamin 1 and 2, respectively, and 41% homology with yeast Dnm1p and Vps1p. Sequencing of several additional clones revealed the presence of two alternatively spliced regions. Northern and western blot analyses showed that this novel dynamin-like protein (DLP1) is ubiquitously expressed in all rat tissues examined.

Immunofluorescence microscopy demonstrated that DLP1 is associated with punctate vesicular structures, many of which extend outward from the perinuclear region along linear arrays partially co-aligning with microtubules. Further double immunofluorescence studies indicated that DLP1-positive spots are not associated with clathrin or conventional dynamin and do not contain endocytosed ligands. Surprisingly, a subpopulation of DLP1-positive spots which are organized in linear arrays co-localize with mitochondria. However, in the immunoblotting of liver subcellular fractions, DLP1 is not enriched in the mitochondrial fraction but fractions of cytoplasmic vesicles and endoplasmic reticulum (ER). ImmunoEM also showed that DLP1 localizes to membrane tubules of the ER.

DLP1 and Dynamin Share Similar Domains

Figure 1: DLP1 Localizes Along Mitochondria. Immunofluorescence double labeling of Clone 9 cells for DLP1 (red) and mitochondria (green) reveals that DLP1-positive structures (A and B, arrows) align along mitochondria. Note while this structural association is convincing, the majority of DLP1-positive structures are cytoplasmic and arranged in linear arrays (arrowheads) where no mitochondria are present. These linear, non-mitochondrial arrays of DLP1 have been shown previously to represent microtubules and ER cisternae. Bar, 2 µm

Function
To test the function of DLP1 in mammalian cells, we made green fluorescent protein (GFP) constructs designed to express either wild type or two point mutations (termed K38A and D231N) in the GTP binding region for expression in Clone 9 cells. Transient expression of wild type GFP-DLP1 revealed punctuate vesicular structures similar to endogenous DLP1. In contrast, the distribution of DLP1 in cells expressing K38A or D231N was clumped or diffuse, respectively.

Cells expressing either mutant showed normal endocytic processes as assessed by LDL and dextran uptake. Interestingly, IF staining of distinct membrane organelles in mutant cells revealed a drastic alteration in ER distribution and a marked aggregation and tubulation of mitochondria. Microinjection of DLP1 antibodies confirmed the phenotypes seen in mutant expression studies. Electron microsocopy of microinjected cells showed a striking change in the morphology and distribution of mitochondria and ER.

Mitochondria are often enlarged and elongated with abnormal internal structures, and many ER cisternae lost discrete structure in the cell periphery and often became swollen and enlarged suggesting that inhibition of DLP1 function leads to drastic morphologic changes in mitochondria and ER. We are currently testing if DLP1 mediates membrane transport of newly synthesized proteins and/or lipids between the ER and mitochondria.

Figure 2: Distribution of DLP1-vesicles coincide with microtubule arrays in cultured cells. Double immunofluorescence microscopy of cultured human fibroblasts (A and B) and Clone 9 cells (C) with antibodies to DLP1 and a-tubulin. There is a high degree of overlap (arrows) between the distribution of DLP1-stained vesicles (A) and the microtubule array (B). Scale bar: 20um. (C) A clone 9 cell stained for DLP! (red) and microtubules (green), showing DLP1-vesicles aligned along the microtubule network. Insert is a higher magnification image of the boxed region revealing the intamate association between the DLP1 vesicles and microtubules. N: Nucleus. Scale bars: 10 um.

DLP Contact Information
The DLP projects have been mainly driven by Dr. Yisang Yoon. If you would like any additional information, please contact Dr. Yoon.